Inducible methods for repressing gene function

ABSTRACT

Methods for the rapid repression of gene function in eucaryotic cells are disclosed including inducible means for both shutting down a targeted gene&#39;s transcription and rapidly removing a targeted gene&#39;s polypeptide product.

1. BACKGROUND OF THE INVENTION

[0001] Genetics is essentially an approach to understanding biologicalprocesses through the systematic elimination of gene function.Historically, the genetic approach has involved the development of“screens” for mutations in genes which affect a specific phenotypictrait of an organism. The great advantage of this approach has been thatno prior knowledge of the molecular nature of the genes involved isrequired because the “screen” identifies the affected genes by markingthem with mutations. The mutation involved is frequently a change in thegene's sequence which results in a loss-of-function of the encoded geneproduct. Unfortunately the genetic approach has many limitations. Indeedthe study of essential genes, required for cell viability, isexceedingly difficult using a purely genetic approach. As the famousmolecular biologist David Botstein once put it, “death is not aphenotype;” an expression which encapsulates the frustration ofattempting to study the function of essential genes. The implementationof “reverse genetics” in yeast (see e.g. Winston et al. (1983) MethodsEnzymol 101: 211-28) and, later, in mammals (see e.g. Capecchi (1989)Science 244: 1288-92), has allowed the positive identification of a geneas essential through the inability to recover viable yeast haploid gene“knockout” spores or homozygous recessive “knockout” mice. Nevertheless,the exact biological processes in which the essential gene is involvedare difficult to determine due to the inability to isolate and/or studythe doomed knockout yeast spore or the inviable homozygous mouse zygote.Thus the downstream effects on specific aspects of cell functionfollowing removal of the essential gene product cannot be readilydetermined using these traditional “knockout” studies. Furthermore,while traditional gene “knockout” experiments may be useful indemonstrating that a given gene is essential for the life of theorganism, they provide no data on precisely how important the gene is orto what extent so-called “second-site suppressing” mutations can arisewhich restore cell viability following the removal of the essentialgene. These considerations are important in the selection of targets forthe rational design of, for example, antibiotic or chemotherapeuticpharmaceutical agents.

[0002] Others have endeavored to devise systems for the directedinactivation of a specific target gene in a host eucaryotic cell. Forexample, in an attempt to provide for a systematic means of derivingtemperature-sensitive conditional alleles of a given gene target, Dohmenet al. have devised a temperature-sensitive “degron” cassette that canbe appended to any gene of interest and used to render itthermosensitive (Dohmen et al. (1994) Science 263: 1273-6). Thisapproach could thus be applied in theory to any essential gene ofinterest. However, the generality with which the thermosensitive degroncan be successfully applied to specific gene targets has yet to bedetermined and the necessity of relying upon thermal induction for theresulting system is a major drawback. Indeed, eucaryotic cellsexperience a transient heat-shock response which can have profoundeffects on some cellular processes such as transcription. Furthermore,the requirement for induction by heat shock precludes useful applicationto mammalian transgenic animal systems. Still other systems have beendeveloped for the specific targeted removal of a host gene. Notably theCre/lox system (see e.g. Sauer (1998) Mehods 14: 381-92) allows for theinducible deletion of a specific target gene through the action of theCre site-specific DNA recombinase. Using this system, genetic switchescan be designed to target ablation of a target gene in a specific tissueand at a specific time during development. One shortcoming of thismethod is that, following recombinational deletion of the targeted genefrom the chromosome, the remaining mRNA and polypeptide products of thegene may only slowly be titrated out of the host cell throughconsecutive mitotic cell divisions and/or the eventual turnover of themRNA and polypeptide by cellular ribonucleases and proteases. Thus itwould be desirable to have a more rapid means for directly inactivatingspecific target genes in a host eucaryotic cell.

2. SUMMARY OF THE INVENTION

[0003] In general, the present invention provides a rapid and effectivemeans for inactivating target genes, including target genes involved inimportant biological pathways. The invention also provides a system forthe rapid and reliable repression of gene function regardless of whetherthe gene of interest is known or suspected of being an essential gene.

[0004] In one aspect, the present invention provides multiple means forthe rapid and inducible elimination of gene function in a controlled andreproducible manner in a population of otherwise mitotically viableeucaryotic cell. The methods described include a method for rapidlyrepressing the transcription of a target gene through the action of aninducible repressor, a method for rapidly removing the polypeptideproduct of a target gene through directed proteolysis, and an integratedmethod in which both transcriptional repression and directed proteolysisoccurs. In one embodiment, the method provides an inducible means forthe passive removal of an mRNA product of a target gene (i.e. new targetgene mRNA synthesis is blocked and the existing target gene mRNA isallowed to degrade through the natural turnover of the remaining targetmRNA). In another embodiment, the method provides an inducible means forthe active removal of a polypeptide product of a target gene (i.e. whilenew target gene polypeptide synthesis, or translation, is not blockedper se, the existing target gene polypeptide product is activelydegraded by proteolysis). In yet another, preferred embodiment, thefirst and second embodiments are “integrated” thereby allowing anoptimal rate at which gene function can be eliminated by thesimultaneous removal of both mRNA and polypeptide products of the gene.

[0005] The present invention thus provides a method of determining whichgenes represent effective targets for the design of antibiotic and/orchemotherapeutic agents. In particular, an array of essential genes canbe screened to determine which are most vital to cell viability usingthe method of the invention. For example, essential genes which, whentargeted for destruction by this two-pronged inducible repressionsystem, result in the immediate death of the host cell, are likely to beeffective targets for antibiotic or chemotherapeutic agents designed tostop cell growth. The present invention further provides a means ofgenetically modifying a population of cells so as to render them subjectto killing by a normally benign inducing agent. This modificationprovides a convenient way to terminate or attenuate the physiologicaleffects on a host organism of a population of bioengineered cells whichhave been delivered to the host. In this application, virtually anyessential gene can be targeted for the inducible repressional shut-offof the present invention. In still other applications, a bioengineeredcell population which produces a specific physiologically active geneproduct could be designed so that the gene product itself is subject tothe inducible repressional shut-off system. When such a bioengineeredcell population is introduced into a host, the delivery of thephysiologically active gene product produced by the bioengineered cellscan be adjusted throughout the lifetime of the host/cell combination byadministration of a benign inducing agent.

3. BRIEF DESCRIPTION OF THE FIGURES

[0006]FIG. 1 provides a generalized illustration of the essentialcomponents of the method for inducible repression of a target gene. Thetwo prongs of the method are illustrated separately here for clarity.Panel A diagrams the essential elements of the first prong of themethod—the inducible transcriptional shut-off of a target gene. Panel Bdiagrams the essential elements of the second prong of the method—theinducible degradation of the target polypeptide.

[0007]FIG. 2 illustrates the manner in which a target gene is modifiedto become newly susceptible to the two prongs of the repression method.FIG. 2A illustrates the replacement of a native target gene promoter(naPr) with a repressible promoter (rePr) which is responsive to aninducible repressor. FIG. 2A thus illustrates the modificationsessential to make the target gene susceptible to the inducibletranscriptional shut-off prong. FIG. 2B illustrates the insertion of aubiquitin coding sequence (Ub) and a unique codon (X), destined tobecome the amino-terminal amino acid residue of the target polypeptide,upstream of the target polypeptide-encoding sequence, or target “ORF.”An optional epitope tag (Ep) marker for the target gene is alsoindicated in the figure. FIG. 2B thus illustrates the modificationsessential to make the target gene susceptible to the inducible N-endrule proteolytic effector.

[0008]FIG. 3 illustrates the generalized structure of a target genewhich has been modified so as to made susceptible to the action of boththe transcriptional shut-off prong and the directed proteolysis prong ofthe method. This preferred embodiment of the modified target gene can bereadily transplaced into the eucaryotic genome at the site of the nativetarget gene by standard “knock-in” technology as shown in Panel B.

4. DETAILED DESCRIPTION OF THE INVENTION

[0009] 4.1 General

[0010] The two prongs of the method are illustrated separately in FIG. 1for clarity, but it is understood that, in a preferred embodiment, thetwo prongs are employed simultaneously to maximize the speed andefficacy of the method. Panel A diagrams the essential elements of thefirst prong of the method—the inducible transcriptional shut-off of atarget gene. Panel B diagrams the essential elements of the second prongof the method—the inducible degradation of the target polypeptide. Bothprongs rely on the use of an inducible promoter (inPr) to drive theinducible expression of an effector of suppression. In Panel A, theeffector of suppression is a transcriptional repressor which is capableof repressing transcription from a repressible promoter (rePr). In panelB, the effector of suppression is a component of the N-end rule pathwaywhich effects the proteolytic destruction of polypeptides possessingcertain amino-terminal amino acid residues. Both prongs further rely onthe construction of a synthetic version of the target gene which hasbeen engineered from its native form so as to be uniquely sensitized tothe effectors of suppression described above. As shown in FIG. 2A, thetarget gene can be modified so that its native promoter is replaced by anatural or synthetic promoter (rePr) which is subject to repression bythe inducible repressor. This alteration of the target gene potentiatesrepression by the transcriptional shut-off prong of the method. As shownin FIG. 2B, the target gene can also be modified so that its encodedpolypeptide (the target ORF—target open reading frame) is fused in frameto the carboxy-terminal codon of a ubiquitin-encoding sequence.Furthermore a unique amino-terminal amino acid encoding codon isengineered at the point of fusion of the two coding sequences—i.e. justdownstream of ubiquitin's final glycine codon and just before the targetORF.

[0011] 4.2 Definitions

[0012] The term “agonist”, as used herein, is meant to refer to an agentthat mimics or upregulates (e.g. potentiates or supplements) bioactivityof the protein of interest. An agonist can be a wild-type protein orderivative thereof having at least one bioactivity of the wild-typeprotein. An agonist can also be a compound that upregulates expressionof a gene or which increases at least one bioactivity of a protein. Anagonist can also be a compound which increases the interaction of apolypeptide of interest with another molecule, e.g, a target peptide ornucleic acid.

[0013] “Antagonist” as used herein is meant to refer to an agent thatdownregulates (e.g. suppresses or inhibits) bioactivity of the proteinof interest. An antagonist can be a compound which inhibits or decreasesthe interaction between a protein and another molecule, e.g., a targetpeptide, such as interaction between ubiquitin and its substrate. Anantagonist can also be a compound that downregulates expression of thegene of interest or which reduces the amount of the wild type proteinpresent.

[0014] The term “allele”, which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide, or several nucleotides, and caninclude substitutions, deletions, and/or insertions of nucleotides. Anallele of a gene can also be a form of a gene containing mutations.

[0015] The term “cell death” or “necrosis”, is a phenomenon when cellsdie as a result of being killed by a toxic material, or otherextrinsically imposed loss of function of a particular essential genefunction.

[0016] “Biological activity” or “bioactivity” or “activity” or“biological function”, which are used interchangeably, for the purposesherein means a catalytic, effector, antigenic or molecular taggingfunction that is directly or indirectly performed by the polypeptides ofthis invention (whether in its native or denatured conformation), or byany subsequence thereof.

[0017] As used herein the term “bioactive fragment of a polypeptide”refers to a fragment of a full-length polypeptide, wherein the fragmentspecifically agonizes (mimics) or antagonizes (inhibits) the activity ofa wild-type polypeptide. The bioactive fragment preferably is a fragmentcapable of interacting with at least one other molecule, protein or DNA,with which a full length protein can bind.

[0018] “Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

[0019] A “chimeric polypeptide” or “fusion polypeptide” is a fusion of afirst amino acid sequence encoding one of the subject polypeptides witha second amino acid sequence defining a domain (e.g. polypeptideportion) foreign to and not substantially homologous with any domain ofa polypeptide. A chimeric polypeptide may present a foreign domain whichis found (albeit in a different polypeptide) in an organism which alsoexpresses the first polypeptide, or it may be an “interspecies”,“intergenic”, etc. fusion of polypeptide structures expressed bydifferent kinds of organisms.

[0020] The term “nucleotide sequence complementary to the nucleotidesequence set forth in SEQ ID NO. x” refers to the nucleotide sequence ofthe complementary strand of a nucleic acid strand having SEQ ID NO. x.The term “complementary strand” is used herein interchangeably with theterm “complement”. The complement of a nucleic acid strand can be thecomplement of a coding strand or the complement of a non-coding strand.

[0021] A “delivery complex” shall mean a targeting means (e.g. amolecule that results in higher affinity binding of a gene, protein,polypeptide or peptide to a target cell surface and/or increasedcellular or nuclear uptake by a target cell). Examples of targetingmeans include: sterols (e.g. cholesterol), lipids (e.g. a cationiclipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associatedvirus, and retrovirus) or target cell specific binding agents (e.g.ligands recognized by target cell specific receptors). Preferredcomplexes are sufficiently stable in vivo to prevent significantuncoupling prior to internalization by the target cell. However, thecomplex is cleavable under appropriate conditions within the cell sothat the gene, protein, polypeptide or peptide is released in afunctional form.

[0022] As is well known, genes or a particular polypeptide may exist insingle or multiple copies within the genome of an individual. Suchduplicate genes may be identical or may have certain modifications,including nucleotide substitutions, additions or deletions, which allstill code for polypeptides having substantially the same activity.Moreover, certain differences in nucleotide sequences may exist betweenindividual organisms, which are called alleles. Such allelic differencesmay or may not result in differences in amino acid sequence of theencoded polypeptide yet still encode a polypeptide with the samebiological activity.

[0023] The terms “epitope” and “epitope tag”, as used herein, are meantto refer to any of various convenient molecular markers known in theart, such as hemagluttinin or FLAG, so that the level of a polypeptidecan be confirmed in a Western blot using, for example, a suitableanti-flu or anti-FLAG antibody.

[0024] The term “equivalent” is understood to include nucleotidesequences encoding functionally equivalent polypeptides of orfunctionally equivalent peptides having an activity of an ---proteinsuch as described herein. Equivalent nucleotide sequences will includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants; and will, therefore, includesequences that differ from the nucleotide sequence of the ---gene shownin SEQ ID NOs:, due to the degeneracy of the genetic code.

[0025] As used herein, the terms “gene”, “recombinant gene” and “geneconstruct” refer to a nucleic acid comprising an open reading frameencoding a polypeptide of the present invention, including both exon and(optionally) intron sequences.

[0026] A “recombinant gene” refers to nucleic acid encoding apolypeptide and comprising—encoding exon sequences, though it mayoptionally include intron sequences which are derived from, for example,a chromosomal gene or from an unrelated chromosomal gene. The term“intron” refers to a DNA sequence present in a given gene which is nottranslated into protein and is generally found between exons.

[0027] “Homology” or “identity” or “similarity” refers to sequencesimilarity between two peptides or between two nucleic acid molecules,with identity being a more strict comparison. Homology and identity caneach be determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. A degree of homology or similarity oridentity between nucleic acid sequences is a function of the number ofidentical or matching nucleotides at positions shared by the nucleicacid sequences. A degree of identity of amino acid sequences is afunction of the number of identical amino acids at positions shared bythe amino acid sequences. A degree of homology or similarity of aminoacid sequences is a function of the number of amino acids, i.e.structurally related, at positions shared by the amino acid sequences.An “unrelated” or “non-homologous” sequence shares less than 40%identity, though preferably less than 25% identity, with one of thesequences of the present invention.

[0028] The term “interact” as used herein is meant to include detectableinteractions (e.g. biochemical interactions) between molecules, such asinteraction between protein-protein, protein-nucleic acid, nucleicacid-nucleic acid, and protein-small molecule or nucleic acid-smallmolecule in nature.

[0029] The term “isolated” as used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject polypeptides preferably includes no more than 10 kilobases (kb)of nucleic acid sequence which naturally immediately flanks the gene ingenomic DNA, more preferably no more than 5 kb of such naturallyoccurring flanking sequences, and most preferably less than 1.5 kb ofsuch naturally occurring flanking sequence. The term isolated as usedherein also refers to a nucleic acid or peptide that is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Moreover, an “isolated nucleicacid” is meant to include nucleic acid fragments which are not naturallyoccurring as fragments and would not be found in the natural state. Theterm “isolated” is also used herein to refer to polypeptides which areisolated from other cellular proteins and is meant to encompass bothpurified and recombinant polypeptides.

[0030] The term “modulation” as used herein refers to both upregulation(i.e., activation or stimulation (e.g., by agonizing or potentiating))and downregulation (i.e. inhibition or suppression (e.g., byantagonizing, decreasing or inhibiting)).

[0031] The term “mutated gene” refers to an allelic form of a gene,which is capable of altering the phenotype of a subject having themutated gene relative to a subject which does not have the mutated gene.If a subject must be homozygous for this mutation to have an alteredphenotype, the mutation is said to be recessive. If one copy of themutated gene is sufficient to alter the genotype of the subject, themutation is said to be dominant. If a subject has one copy of themutated gene and has a phenotype that is intermediate between that of ahomozygous and that of a heterozygous subject (for that gene), themutation is said to be co-dominant.

[0032] The “non-human animals” of the invention include mammalians suchas rodents, non-human primates, sheep, dog, cow, chickens, amphibians,reptiles, etc. Preferred non-human animals are selected from the rodentfamily including rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding andidentifying agents which can affect, for example, embryogenesis andtissue formation. The term “chimeric animal” is used herein to refer toanimals in which the recombinant gene is found, or in which therecombinant gene is expressed in some but not all cells of the animal.The term “tissue-specific chimeric animal” indicates that one of therecombinant gene is present and/or expressed or disrupted in sometissues but not others.

[0033] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0034] As used herein, the term “promoter” means a DNA sequence thatregulates expression of a selected DNA sequence operably linked to thepromoter, and which effects expression of the selected DNA sequence incells. The term encompasses “tissue specific” promoters, i.e. promoters,which effect expression of the selected DNA sequence only in specificcells (e.g. cells of a specific tissue). The term also covers so-called“leaky” promoters, which regulate expression of a selected DNA primarilyin one tissue, but cause expression in other tissues as well. The termalso encompasses non-tissue specific promoters and promoters thatconstitutively express or that are inducible (i.e. expression levels canbe controlled).

[0035] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a gene product.

[0036] The term “recombinant protein” refers to a polypeptide of thepresent invention which is produced by recombinant DNA techniques,wherein generally, DNA encoding a -- polypeptide is inserted into asuitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant -- gene, is meant to include withinthe meaning of “recombinant protein” those proteins having an amino acidsequence of a native -- polypeptide, or an amino acid sequence similarthereto which is generated by mutations including substitutions anddeletions (including truncation) of a naturally occurring form of thepolypeptide.

[0037] The term “repression” as used herein is meant to include“inducible repression” and is used to refer to transcriptionalrepression as by a transcriptional repressor such as a DNA bindingtranscriptional repressor which binds a target promoter (a “repressible”promoter) to be repressed.

[0038] The term “degrading” as used herein is meant to include“inducible degradation” and is used to refer to proteolytic degradationas may be facilitated by a component of the N-end rule proteolyticpathway. Such an “inducible degradation,” as referred to herein, ismeant to describe the targeted degradation of a specific “target genepolypeptide.”

[0039] “Small molecule” as used herein, is meant to refer to acomposition, which has a molecular weight of less than about 5 kD andmost preferably less than about 4 kD. Small molecules can be nucleicacids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids orother organic (carbon containing) or inorganic molecules. Manypharmaceutical companies have extensive libraries of chemical and/orbiological mixtures, often fungal, bacterial, or algal extracts, whichcan be screened with any of the assays of the invention to identifycompounds that modulate a bioactivity.

[0040] “Transcription” is a generic term used throughout thespecification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked.“Transcriptional repressor,” as used herein, refers to any of variouspolypeptides of procaryotic, eucaryotic origin, or which are syntheticartificial chimeric constructs, capable of repression either alone or inconjunction with other polypeptides and which repress transcription ineither an active or a passive manner as described elsewhere. It willalso be understood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring forms of polypeptide.

[0041] As used herein, the term “transfection” means the introduction ofa nucleic acid, e.g., via an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a polypeptide or, inthe case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the polypeptide isdisrupted.

[0042] As used herein, the term “target gene” refers to the nucleic acidwhich encodes a gene of interest. The target gene can be an “essential”gene, required for continued cell viability whose function is to beshut-off by the method of the invention. The term “target gene” is usedto refer to both the original gene to be targeted for shut-off and thesame gene as later modified for shut-off (such as by the replacement ofthe native promoter with a repressible promoter and the addition of aubiquitin-X encoding sequence to the amino terminus of the targeted ORF,or open reading frame). The term “target polypeptide” is usedinterchangeably with the term “target gene polypeptide” and refers tothe polypeptide gene product of the target gene as described above.

[0043] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., one of the polypeptides, or an antisensetranscript thereto) which has been introduced into a cell. A transgenecould be partly or entirely heterologous, i.e., foreign, to thetransgenic animal or cell into which it is introduced, or, homologous toan endogenous gene of the transgenic animal or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe animal's genome in such a way as to alter the genome of the cellinto which it is inserted (e.g., it is inserted at a location whichdiffers from that of the natural gene or its insertion results in aknockout). A transgene can also be present in a cell in the form of anepisome. A transgene can include one or more transcriptional regulatorysequences and any other nucleic acid, such as introns, that may benecessary for optimal expression of a selected nucleic acid.

[0044] A “transgenic animal” refers to any animal, preferably anon-human mammal, bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by transgenic techniques well known inthe art. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of one of the polypeptide, e.g. either agonistic or antagonisticforms. However, transgenic animals in which the recombinant -- gene issilent are also contemplated, as for example, the FLP or CRE recombinasedependent constructs described below. Moreover, “transgenic animal” alsoincludes those recombinant animals in which gene disruption of one ormore genes is caused by human intervention, including both recombinationand antisense techniques.

[0045] The term “treating” as used herein is intended to encompasscuring as well as ameliorating at least one symptom of the condition ordisease.

[0046] The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof preferred vector is an episome, i.e., a nucleic acid capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of “plasmids” which refer generally tocircular double stranded DNA loops which, in their vector form are notbound to the chromosome. In the present specification, “plasmid” and“vector” are used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors which serve equivalent functions andwhich become known in the art subsequently hereto.

[0047] The term “wild-type allele” refers to an allele of a gene which,when present in two copies in a subject results in a wild-typephenotype. There can be several different wild-type alleles of aspecific gene, since certain nucleotide changes in a gene may not affectthe phenotype of a subject having two copies of the gene with thenucleotide changes.

[0048] The term “ubiquitin” as used herein refers to an abundant 76amino acid residue polypeptide that is found in all eukaryotic cells.The ubiquitin polypeptide is characterized by a carboxy-terminal glycineresidue that is activated by ATP to a high-energy thiol-esterintermediate in a reaction catalyzed by a ubiquitin-activating enzyme(E1). The activated ubiquitin is transferred to a substrate polypeptidevia an isopeptide bond between the activated carboxy-terminus ofubiquitin and the epsilon-amino group of a lysine residue(s) in theprotein substrate. This transfer requires the action of ubiquitinconjugating enzymes such as E2 and, in some instances, E3 activities.The ubiquitin modified substrate is thereby altered in biologicalfunction, and, in some instances, becomes a substrate for components ofthe ubiquitin-dependent proteolytic machinery which includes bothubiquitin isopeptidase enzymes as well as proteolytic proteins which aresubunits of the proteasome. As used herein, the term “ubiquitin”includes within its scope all known as well as unidentified eukaryoticubiquitin homologs of vertebrate or invertebrate origin. Examples ofubiquitin polypeptides as referred to herein include the human ubiquitinpolypeptide which is encoded by the human ubiquitin encoding nucleicacid sequence (GenBank Accession Numbers: U49869, X04803) as well as allequivalents. Equivalent ubiquitin polypeptide encoding nucleotidesequences are understood to include those sequences that differ by oneor more nucleotide substitutions, additions or deletions, such asallelic variants; as well as sequences which differ from the nucleotidesequence encoding the human ubiquitin coding sequence shown in SEQ IDNO. 2, due to the degeneracy of the genetic code. Another example of aubiquitin polypeptide as referred to herein is murine ubiquitin which isencoded by the murine ubiquitin encoding nucleic acid sequence (GenBankAccession Number: X51730).

[0049] The term “ubiquitin mutants” as used herein refers to naturallyoccurring and synthetically derived altered forms of the ubiquitinpolypeptide molecule described above. Such mutants include polypeptidesencoded by ubiquitin nucleic acid coding sequences containing missensemutations, which produce altered amino acid sequences at a specificresidue(s), and nonsense mutations which produce STOP codons resultingin the formation of truncated polypeptide. These mutations also includeinsertions and deletions which produce frame-shifts or amino acidresidue insertions and deletions. These mutants thus produce alteredcoding sequences resulting in the synthesis of altered forms of theubiquitin polypeptide other than those described by the term “ubiquitin”as defined above. Examples of ubiquitin mutants described herein includethe Ub-75 polypeptide and Ub(K48R). In the Ub-75 polypeptide, SEQ ID NO.3, the carboxy-terminal glycine codon of ubiquitin (SEQ ID NO. 2) isreplaced by a stop codon resulting in the synthesis of a mutantubiquitin polypeptide characterized by a carboxy-terminal glycineresidue corresponding to the penultimate glycine residue of wild typeubiquitin. Ub(K48R) is a mutant in which the 48^(th) residue of wildtype ubiquitin, which corresponds to a lysine residue used in apolyubiquitination cross-linking reaction, is changed to an arginineresidue which cannot accommodate the polyubiquitination cross-linkingreaction.

[0050] The term “ubiquitin-like protein” as used herein refers to agroup of naturally occurring proteins, not otherwise describable asubiquitin equivalents, but which nonetheless show strong amino acidhomology to ubiquitin. As used herein this term includes thepolypeptides NEDD8, UBL1, NPVAC, and NPVOC. These “ubiquitin-likeproteins” are at least over 40% identical in sequence to the humanubiquitin polypeptide and contain a pair of carboxy-terminal glycineresidues which function in the activation and transfer of ubiquitin totarget substrates as described supra.

[0051] As used herein, the term “ubiquitin-related protein” as usedherein refers to a group of naturally occurring proteins, not otherwisedescribable as ubiquitin equivalents, but which nonetheless show somerelatively low degree (<40% identity) of amino acid homology toubiquitin. These “ubiquitin-related” proteins include human UbiquitinCross-Reactive Protein (UCRP, 36% identical to huUb, Accession No.P05161), FUBI (36% identical to huUb, GenBank Accession No. AA449261),and Sentrin/Sumo/Pic1 (20% identical to huUb, GenBank Accession No.U83117). The term “ubiquitin-related protein” as used herein furtherpertains to polypeptides possessing a carboxy-terminal pair of glycineresidues and which function as protein tags through activation of thecarboxy-terminal glycine residue and subsequent transfer to a proteinsubstrate.

[0052] The term “ubiquitin-homologous protein” as used herein refers toa group of naturally occurring proteins, not otherwise describable asubiquitin equivalents or ubiquitin-like or ubiquitin-related proteins,which appear functionally distinct from ubiquitin in their ability toact as protein tags, but which nonetheless show some degree of homologyto ubiquitin (34-41% identity). These “ubiquitin-homologous proteins”include RAD23A (36% identical to huUb, SWISS-PROT. Accession No.P54725), RAD23B (34% identical to huUb, SWISS-PROT. Accession No.P54727), DSK2 (41% identical to huUb, GenBank Accession No. L40587), andGDX (41% identical to huUb, GenBank Accession No. J03589). The term“ubiquitin-homologous protein” as used herein is further meant tosignify a class of ubiquitin homologous polypeptides whose similarity toubiquitin does not include glycine residues in the carboxy-terminal andpenultimate residue positions. Said proteins appear functionallydistinct from ubiquitin, as well as ubiquitin-like and ubiquitin-relatedpolypeptides, in that, consistent with their lack of a conservedcarboxy-terminal glycine for use in an activation reaction, they havenot been demonstrated to serve as tags to other proteins by covalentlinkage.

[0053] The term “ubiquitin conjugation machinery” as used herein refersto a group of proteins which function in the ATP-dependent activationand transfer of ubiquitin to substrate proteins. The term thusencompasses: E1 enzymes, which transform the carboxy-terminal glycine ofubiquitin into a high energy thiol intermediate by an ATP-dependentreaction; E2 enzymes (the UBC genes), which transform the E1-S˜Ubiquitinactivated conjugate into an E2-S˜Ubiquitin intermediate which acts as aubiquitin donor to a substrate, another ubiquitin moiety (in apoly-ubiquitination reaction), or an E3; and the E3 enzymes (orubiquitin ligases) which facilitate the transfer of an activatedubiquitin molecule from an E2 to a substrate molecule or to anotherubiquitin moiety as part of a polyubiquitin chain. The term “ubiquitinconjugation machinery”, as used herein, is further meant to include allknown members of these groups as well as those members which have yet tobe discovered or characterized but which are sufficiently related byhomology to known ubiquitin conjugation enzymes so as to allow anindividual skilled in the art to readily identify it as a member of thisgroup. The term as used herein is meant to include novel ubiquitinactivating enzymes which have yet to be discovered as well as thosewhich function in the activation and conjugation of ubiquitin-like orubiquitin-related polypeptides to their substrates and topoly-ubiquitin-like or polyubiquitin-related protein chains.

[0054] The term “ubiquitin-dependent proteolytic machinery” as usedherein refers to proteolytic enzymes which function in the biochemicalpathways of ubiquitin, ubiquitin-like, and ubiquitin-related proteins.Such proteolytic enzymes include the ubiquitin C-terminal hydrolases,which hydrolyze the linkage between the carboxy-terminal glycine residueof ubiquitin and various adducts; ubiquitin isopeptidases, whichhyrolyze the glycine76-lysine48 linkage between cross-linked ubiquitinmoieties in poly-ubiquitin conjugates; as well as other enzymes whichfunction in the removal of ubiquitin conjugates from ubiquitinatedsubstrates (generally termed “deubiquitinating enzymes”). Theaforementioned protease activities function in the removal of ubiquitinunits from a ubiquitinated substrate following or duringubiquitin-dependent degradation as well as in certain proofreadingfunctions in which free ubiquitin polypeptides are removed fromincorrectly ubiquitinated proteins. The term “ubiquitin-dependentproteolytic machinery” as used herein is also meant to encompass theproteolytic subunits of the proteasome (including human proteasomesubunits C2, C3, C5, C8, and C9). The term “ubiquitin-dependentproteolytic machinery” as used herein thus encompasses two classes ofproteases: the deubiquitinating enzymes and the proteasome subunits. Theprotease functions of the proteasome subunits are not known to occuroutside the context of the assembled proteasome, however independentfunctioning of these polypeptides has not been excluded.

[0055] The term “ubiquitin system” as referred to herein is meant todescribe all of the aforementioned components of the ubiquitinbiochemical pathways including ubiquitin, ubiquitin-like proteins,ubiquitin-related proteins, ubiquitin-homologous proteins, ubiquitinconjugation machinery, ubiquitin-dependent proteolytic machinery, or anyof the substrates which these ubiquitin system components act upon.

[0056] 4.3 Essential Components of the Method

[0057] The following sections describe in detail various alternativeembodiments of each of the elements of the general methods describedabove. In particular, section 4.3.1 describes in detail one necessarycomponent, an inducible promoter, which is useful for either of the twoprongs of the method. Furthermore section 4.3.5 provides a descriptionof the various target genes which can be employed in any of therepression methods. Section 4.3.5 also describes methods for modifyingthe existing native target gene to make it responsive to either or bothprongs of the repression system. Section 4.3.2 describes in detail thetranscriptional repressors and corresponding repressible promoters whichare essential to the design of the transcriptional repression prong ofthe method. Sections 4.3.3 and 4.3.4 describe in detail the componentsessential for the polypeptide degradation prong of the method. Inparticular, section 4.3.3 describes various embodiments of the N-endrule gene which is inducibly deployed to effect proteolysis of thetarget polypeptide. Section 4.3.4 describes the ubiquitin and ubiquitinequivalent sequences which are used to produce, through endoproteolyticprocessing of a ubiquitin-target polypeptide fusion protein, targetpolypeptides possessing unique (generally non-methionine) amino-terminalamino acids which subject the target polypeptide to N-end ruleproteolytic processes.

[0058] 4.3.1 Inducible Promoters

[0059] In both prongs of the method of the present invention, aninducible promoter is employed to drive expression of the “effector ofsuppression.” Thus in the inducible transcriptional repression prong ofthe method the inducible promoter is used to drive expression of thetranscriptional repressor, while in the inducible proteolyticdegradation prong of the method the inducible promoter is used to driveexpression of the N-end rule gene (see FIGS. 1A and 1B). In a preferredmode of the invention, identical or unique inducible promoters are usedto drive the independent or coupled expression of both a transcriptionalrepressor and an N-end rule gene. The inducible promoters of the presentinvention are capable of functioning in a eucaryotic host organism.Preferred embodiments include naturally occurring yeast and mammalianinducible promoters as well as synthetic promoters designed to functionin a eucaryotic host as described below. The important functionalcharacteristic of the inducible promoters of the present invention istheir ultimate inducibility by exposure to an environmental inducingagent. Appropriate environmental inducing agents include exposure toheat, various steroidal compounds, divalent cations (including Cu⁺² andZn⁺²), galactose, tetracycline, IPTG (isopropyl β-D thiogalactoside), aswell as other naturally occurring and synthetic inducing agents andgratuitous inducers. It is important to note that, in certain modes ofthe invention, the environmental inducing signal can correspond to theremoval of any of the above listed agents which are otherwisecontinuously supplied in the uninduced state (see the tTA based systemdescribed below for example). The inducibility of a eucaryotic promotercan be achieved by either of two mechanisms included in the method ofthe present invention. Suitable inducible promoters can be dependentupon transcriptional activators which, in turn, are reliant upon anenvironmental inducing agent. Alternatively the inducible promoters canbe repressed by a transcriptional repressor which itself is renderedinactive by an environmental inducing agent. Thus the inducible promotercan be either one which is induced by an environmental agent whichpositively activates a transcriptional activator, or one which isderepressed by an environmental agent which negatively regulates atranscriptional repressor. We note here that the latter class ofinducible promoter systems defines transcriptional repressors andcorresponding negative cis regulatory elements which can also find useas the repressors and corresponding repressible promoters of the presentinvention as described in section 4.3.2.

[0060] The inducible promoters of the present invention include thosecontrolled by the action of latent transcriptional activators which aresubject to induction by the action of environmental inducing agents.Preferred examples include the copper inducible promoters of the yeastgenes CUP1, CRS5, and SOD1 which are subject to copper-dependentactivation by the yeast ACE1 transcriptional activator (see e.g. Strainand Culotta (1996) Mol Gen Genet 251: 139-45; Hottiger et al. (1994)Yeast 10: 283-96; Lapinskas et al. (1993) Curr Genet 24: 388-93; andGralla et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8558-62).Alternatively, the copper inducible promoter of the yeast gene CTT1(encoding cytosolic catalase T), which operates independently of theACE1 transcriptional activator (Lapinskas et al. (1993) Curr Genet 24:388-93), can be utilized. The copper concentrations required foreffective induction of these genes are suitably low so as to betolerated by most cell systems, including yeast and Drosophila cells.Alternatively, other naturally occurring inducible promoters can be usedin the present invention including: steroid inducible gene promoters(see e.g. Oligino et al. (1998) Gene Ther. 5: 491-6); galactoseinducible promoters from yeast (see e.g. Johnston (1987) Microbiol Rev51: 458-76; Ruzzi et al. (1987) Mol Cell Biol 7: 991-7); and variousheat shock gene promoters. Many eucaryotic transcriptional activatorshave been shown to function in a broad range of eucaryotic host cells,and so, for example, many of the inducible promoters identified in yeastcan be adapted for use in a mammalian host cell as well. For example, aunique synthetic transcriptional induction system for mammalian cellshas been developed based upon a GAL4-estrogen receptor fusion proteinwhich induces mammalian promoters containing GAL4 binding sites(Braselmann et al. (1993) Proc Natl Acad Sci USA 90: 1657-61). These andother inducible promoters responsive to transcriptional activators whichare dependent upon specific inducing agents are suitable for use withthe present invention.

[0061] The inducible promoters of the present invention also includethose which are repressed by repressors which are subject toinactivation by the action of environmental inducing agents. Examplesinclude procaryotic repressors which can transcriptionally represseucaryotic promoters which have been engineered to incorporateappropriate repressor-binding operator sequences. Preferred repressorsfor use in the present invention are sensitive to inactivation byphysiologically benign inducing agent. Thus, where the lac repressorprotein is used to control the expression of a eucaryotic promoter whichhas been engineered to contain a lacO operator sequence, treatment ofthe host cell with IPTG will cause the dissociation of the lac repressorfrom the engineered promoter and allow transcription to occur.Similarly, where the tet repressor is used to control the expression ofa eucaryotic promoter which has been engineered to contain a tetOoperator sequence, treatment of the host cell with IPTG will cause thedissociation of the tet repressor from the engineered promoter and allowtranscription to occur.

[0062] In a preferred embodiment of the invention, the repressor of theinducible promoter is synthesized as a ubiquitin fusion proteinconforming to the formula ubiquitin-X-repressor. This can be achievedusing the ubiquitin fusion vector systems designed to confer inducibleproteolytic sensitivity to the target gene polypeptide as describedbelow. Thus it will be appreciated by the skilled artisan that a rapidinduction of a repressible promoter can be achieved by simultaneouslydelivering an environmental inducing agent which causes dissociation ofthe repressor from the repressed inducible promoter, and simultaneouslypromoting the destruction of that repressor by N-end rule directedproteolysis. Degradation of the repressor prevents rebinding to theoperator which can result in decreased inducibility of the repressiblepromoter—a problem which has been recognized in the art (see Gossen etal. (1993) TIBS 18: 471-5). Furthermore, this aspect of the inventioncan be utilized independently of the targeted shut-off of a gene, togenerally increase the inducibility of a eucaryotic expression systemwhich is subject to repression by a repressor. Thus the presentinvention further provides improved methods for inducible expression ofendogenous or heterologous genes in a eucaryotic cell.

[0063] As suggested above, the inducible promoters of the presentinvention include those which are not naturally occurring promoters butrather synthetically derived inducible promoter systems which may makeuse of procaryotic transcriptional repressor proteins. The advantage ofusing prokaryotic repressor proteins in the invention is theirspecificity to a corresponding bacterial operator binding site, whichcan be incorporated into the synthetic inducible promoter system Theseprocaryotic repressor proteins have no natural eucaryotic gene targetsand affect only the effector of suppression gene which is put under thetranscriptional control of the inducible synthetic promoter. This systemthereby avoids undesirable side-effects resulting from unintentionalalteration of the expression of nontargeted eucaryotic genes when theinducible promoter is induced. A preferred example of this type ofinducible promoter system is the tetracycline-regulated induciblepromoter system. Various useful versions of this promoter system havebeen described (see Shockett and Schatz (1996) Proc. Natl. Acad. Sci.USA 93: 5173-76 for review). As suggested above, thesetetracycline-regulated systems generally make use of a strong eucaryoticpromoter, such as human cytomegalovirus (CMV) immediate early (IE)promoter/enhancer and a tet resistance operator (tetO) which is bound bythe tet repressor protein. In a preferred embodiment, the systeminvolves a modified version of the tet repressor protein called areverse transactivator (rtTA, or rtTA-nls, which contains a nuclearlocalization signal) which binds tetO sequences only in the presence ofthe tet derivatives doxycycline or anhydrotetracycline. Using thissystem, a synthetic human CMV/IE-tetO-promoter driven construct could beinduced by 3 orders of magnitude in 20 hrs by the addition of the tetderivatives (see Gossen et al. (1995) Science 268: 1766-9). Thus thissystem can be used to make the effector of suppression genes of thepresent invention inducible in response to the delivery of tetracyclinederivatives to the targeted eucaryotic cell. Alternatively, a tetrepressor fused to a transcriptional activation domain of VP 16 (tTA)can be used to drive expression of the inducible promoter of the presentinvention In this instance, transcriptional activation of a synthetichuman CMV/IE-tetO-promoter driven construct is achieved by the removalof tetracycline since the tTA activator only binds to the tetO in theabsence of tet (see Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA89: 5547-51). Other synthetic inducible promoter systems are alsoavailable for use in the present invention. For example, a lacrepressor-VP 16 fusion which exhibits a “reverse” DNA binding phenotype(i.e., analogous to rtTA described above, it only binds the lacOoperator sequence in the presence of the inducer IPTG) (see Lambowitzand Belfort (1993) Annu Rev Biochem 62: 587-622). This particularsynthetic inducible promoter is approximately 1000-fold inducible in thepresence of IPTG. Since neither the tet repressor gene nor the lacrepressor gene occurs naturally in a eucaryotic cell, systems involvingsynthetic inducible promoter constructs such as these rely on thefurther delivery of an expressible copy of the appropriate procaryoticrepressor gene. Suitable expression cassettes for this purpose arereadily available for heterologous expression in many differenteucaryotic cells including various yeast species and mammalian cells.

[0064] The present invention thus allows for considerable flexibility inchoosing a suitable inducible promoter and corresponding inducing agent.In some embodiments of the invention, the choice of inducible promotermay be governed by the suitability of the required inducing agent.Factors such as cytotoxicity or indirect effects on nontarget genes maybe important to consider in this instance. In other instances the choicemay be governed by the properties of the inducible system as a whole. Inparticular, the ease with which the system can be introduced into theappropriate host cell and the speed and strength with which induction ofthe system occurs following exposure to an inducing agent.

[0065] As mentioned above, the inducible promoters of the presentinvention are used to drive expression of the effector of suppressiongenes utilized in each of the two prongs of the method of the presentinvention (see FIGS. 1A & 1B). These effector of suppression genesinclude transcriptional repressors (described below) and N-end rulesystem genes (described in section 4.3.3).

[0066] 4.3.2 Repressors and Corresponding Repressible Promoters

[0067] Although these two elements—the transcriptional repressor and thecorresponding repressible promoters are understood to be independentlyimplementable by the method of the invention, the choice of one of theseelements governs the selection of the other and so they are discussedtogether here for the sake of convenience. Nonetheless, in preferredembodiments of the invention the two components are separately andindependently engineered into the targeted eucaryotic cell. Inparticular, a host cell engineered to contain an inducibletranscriptional repressor and an inducible N-end rule system component,can be maintained independently for an indefinite period of time priorto the introduction of a target gene construct subject to repression bythe transcriptional repressor. Such a host cell can serve as a “mastercell line” which carries all of the essential components of thetwo-pronged shutoff system. In preferred embodiments this “master cellline” comprises both an inducible repressor of the target gene and aninducible N-end rule system component for proteolytic destruction of thetarget polypeptide. It is understood, however, that in certain instancesa “master cell line” carrying only one or the other inducible “effectorof suppression” of the two-pronged shut-off system will be desired. Forexample, a cell-line containing only the transcriptional shut-off prongmay be used to determine the normal half-life of a target gene. Thecomplete block to target gene transcription in such a case would allowone to follow the rate of degradation of the target polypeptide (forinstance by Western analysis) without having to radioactively label thetarget as in a “pulse-chase” type experiment. The complete master cellline carrying both inducible effectors of suppression is, however, apreferred embodiment of the invention as it allows the creation ofmultiple otherwise “isogenic” cell lines which differ only in thespecific gene which has been engineered to be subject to the inducibletranscriptional/proteolytic shut-off system.

[0068] Suitable repressors and repressible promoters for use with theimmediate invention include those utilizing procaryotic repressors asdiscussed above in the description of suitable inducible promotersystems. However, the requirements for suitability of a repressor foruse as a repressor of the target gene are fewer than are therequirements for a suitable repressor for use in the inducible promotersystem of the invention. In particular, such target gene repressors neednot be inactivatable by an inducing agent. Thus, the lacI repressor orthe tet repressor could be used in this context without regard to theneed to reverse their repression with IPTG or tetracycline (inducingagents). This is because reversal of the transcriptional inactivation ofthe target gene is not generally desired in the present invention. Thusvirtually any site-specific DNA binding protein, which is not otherwisea transcriptional activator and for which a high-affinity bindingsequence is known, is suitable for use as a repressor in the presentinvention. The only requirement is that the high affinity bindingsequence be incorporated into the repressible promoter used to expressthe target gene and that the site-specific DNA binding protein, whenbound to this sequence, is capable of repressing the transcription ofthe target gene. Repression can be achieved by either active or passiveprocesses as is generally understood in the art. For example, theprocaryotic lacI and tet repressors are generally believed to be capableof repression in eucaryotes due to a passive ability to blocktrancription from a eucaryotic promoter to which they are bound(so-called “steric” blocking of the transcription apparatus). Incontrast, active repression occurs when a DNA binding protein recruitsother cellular proteins involved in transcriptional repression. Forexample, in Saccharomyces cerevisiae the Ssn6-Tup1 corepressor isrecruited by a number of different DNA-binding repressor proteins. Theseinclude ROX1, a preferred repressor of the present invention, which isdescribed in detail below. Other systems sensitive to Ssn6-Tup 1repression in yeast include the DIT1 and DIT2 genes in yeast which arerepressed through a cis sequence called NREDIT (Friesen et al. (1998)Genetics 150: 59-73). Negative regulation by the NREDIT sequenceresponds to mutations in SIN4 and ROX3. Thus, in the present invention,ROX3 could be used as a repressor and the DITi or DIT2 promoter (orminimally an heterologous promoter incorporating the NREDIT element) asthe repressible promoter used to drive expression of the target gene.Alternatively, the MIG1 zinc-finger protein, which recruits theSsn6-Tup1 repressor complex to glucose-repressed promoters (Treitel andCarlson (1995) Proc. Natl. Acad. Sci. USA 92: 3132-6), can be used asthe repressor of the present invention in conjunction with a suitableglucose-repressible promoter to drive expression of the target gene.Furthermore, virtually any site specific DNA binding protein can beadapted for use as a repressor in the present invention by fusing theDNA binding polypeptide to a protein domain known to recruit theSsn6-Tup1 complex. For example, the yeast alpha 2 repressor is known torecruit the Ssn6-Tup1 complex and thus the appropriate alpha 2 codingsequence could be fused to virtually and DNA binding polypeptide inorder to derive a suitable repressor protein for use in the presentinvention.

[0069] Many examples of eucaryotic transcriptional repressors which“actively” repress transcription through a specific cis element areknown in the art and are of use in the present invention. Surprisingly,even some eucaryotic transcriptional activators can be converted intoactive repressor complexes when bound with an appropriate corepressorprotein. For example, MCM1 functions as an activator in yeast but theMCM1/alpha 2 complex is an active repressor complex capable ofrepressing a cis-linked promoter (see e.g. Jonson and Herskowitz (1985)Cell 42: 237-47). Similarly, the Drosophila developmental regulatorDorsal is a transcriptional activator which behaves as an activerepressor when bound to certain cis regulatory elements such as the zengene VRE (ventral repression element, see e.g. Pan and Courey (1992)EMBO 11: 1837-42). In these instances either the activator (MCM1 orDorsal for example) or the corepressor with which it acts (alpha 2 orGroucho (Dubnicoff et al. (1997) Genes Dev 11: 2952-7) is suitable foruse in the present invention where a suitable responsive promoterelement (such as STE6 operator or a zen VRE) is available to controltranscription of the target gene.

[0070] In a preferred embodiment, the transcriptional repressor of thepresent invention is ROX1 and the repressible promoter is selected fromthe group consisting of: ANB1, HEM13, ERG11 and OLE1. ROX1 is awell-studied a transcriptional repressor of hypoxic genes (Lowry, C. V.,Cerdan, M. E., and Zitomer, R. (1990) Mol. Cell Biol. 10: pp. 5921-5926;Balasubramanian, B., Lowry, C. V., and Zitomer, R. S. (1993) Mol. CellBiol. 13: pp. 6071-6078). ROX1 binds to specific hypoxic concensussequences located in the upstream of the upstream region of these genesand represses transcription in conjunction with the general repressioncomplex Tup1-Ssn6 (Deckert et al. (1995) Mol Cell Biol 15: 6109-17).When placed under the control of an ACE I-dependent and copper induciblepromoter from the yeast genes CUP1, CRS5, or SOD1,

[0071] the inPr-ROX1 construct can be stably integrated into the yeastgenome, for example, by conventional two-step gene replacement. In theabsence of copper, ROX1 is not expressed. However when copper is addedto the strain, ROX1 is expressed to levels high enough to repress itstarget genes, but not high enough to impair cell growth, as occurs withgalactose-inducible ROX1 constructs (Deckert, J., Perini, R.,Balasubramanian, B., and Zitomer, R. S. (1995) Genetics 139: pp.1149-58).

[0072] In preferred embodiments employing the ROX1 repressor, a 5′fragment of the coding sequence of the target gene of interest can thenbe genetically modified so that the ROX1-repressible promoter, such asthe ANB1 promoter, replaces the native promoter (naPr) of the targetgene as shown in FIG. 2A. This genetic modification can be achieved byeither standard recombinant DNA subcloning manipulations which are knownin the art, or by in vivo homologous cross-over events which occur at arelatively high frequency from double-stranded DNA ends in Saccharomycescerevisiae and which can also be selected for in mammalian systems usinga “double selection” method known in the art. This ANB1 driven allele,when introduced into the inPr-ROX1 parent strain, renders the resultingANB1 driven allele susceptible to repression by the inducing agent ofthe inducible promoter. Where the inducible promoter (inPr) is acopper-inducible promoter such as from CUP1, the resulting host cellwill express the target gene constitutively in the absence of Cu⁺².Addition of Cu⁺² causes the rapid ROX1-dependent transcriptionalrepression of the target gene. In the absence of de novo synthesis ofthe target gene mRNA, the existing pool of target gene mRNA will bedegraded through normal mRNA “turnover” processes. Thus no further denovo target gene polypeptide synthesis can occur and only the remainingpool of target gene polypeptide remains to provide function to the hostcell. The second prong of the repression system is thereforespecifically tied to removing this residual target gene polypeptidethrough a process of inducible targeted proteolysis.

[0073] 4.3.3 N-End Rule Pathway Components and CorrespondingAmino-Terminal Codons

[0074] The second prong of the two-pronged gene shut-off system is aninducible proteolytic means for the degradation of the existing pool oftarget gene polypeptide. In combination with the transcriptionalshut-off described above, it provides for a thorough block to continuedtarget gene function.

[0075] The targeted inducible proteolytic prong of the system makes useof an inducible promoter, as described above, to drive expression of acomponents of the so-called “N-end rule” system for proteolyticdegradation (Bachmair et al. (1986) Science 234: 179-86). This systemoperates to degrade a cellular polypeptide at a rate dependent upon theamino-terminal amino acid residue of that polypeptide. Proteintranslation ordinarily initiates with an ATG methionine codon and somost polypeptides have an amino-terminal methionine residue and aretypically relatively stable in vivo. For example, in the yeast S.cerevisiae, a beta-galactosidase polypeptide with a methionine aminoterminus has a half-life of >20 hours (Varshavsky (1992) Cell 725-35).Under certain circumstances, however, polypeptides possessing anon-methionine amino-terminal residue can be created. For example, whenan endoprotease hydrolyzes and thus cleaves a unique polypeptide bond(Y-X) internal to a polypeptide, it results in the release of twoseparate polypeptides—one of which possesses an amino-terminal aminoacid, X, which may not be methionine. For example, the endoproteaseubiquitin isopeptidase, which is a preferred component of the presentinvention, will cleave a polypeptide bond carboxy-terminal to the finalglycine residue (codon 76), regardless of what the next codon is. In thenormal function of the cell, this isopeptidase serves to cleave apolyubiquitin precursor into individual ubiquitin units. However it canalso be used to generate a target polypeptide with virtually anyamino-terminal residue by merely fusing the target polypeptide in-frameto a codon corresponding to the desired amino-terminal amino acid (X),which codon, in turn, is fused downstream of ubiquitin (typicallycontiguous with ubiquitin Gly codon 76). The resulting target genechimera construct, has the general structure Ubiquitin-X-Target.Preferred target constructs further comprise an epitope tag (Ep) so thatthe resulting target gene chimera construct has the general structureUbiquitin-X-Ep-target, which results in the eventual production of apolypeptide of the general structure X-Ep-Target. Constitutively activeubiquitin isopeptidase activities present in eucaryotic cells willresult in the endoproteolytic processing of the Ubiquitin-X-Targetpolypeptide into Ubiquitin and X-Target entities. The X-Targetpolypeptide is further acted upon by the components of the N-end rulesystem as described below.

[0076] It has been determined, with reasonable reliability, the relativeeffect of a given amino-terminal residue, X, upon target polypeptidestability. For example, when all 20 possible amino-terminal amino acidresidues were tested to determine their effect on the stability ofbeta-galactosidase (utilizing a ubiquitin-X-beta-galactosidase chimericfusion) in Saccharomyces cerevisiae, drastic differences were discovered(see Varshavsky (1992) Cell 69: 725-35). For example when X was met,cys, ala, ser, thr, gly, val, or pro, the resulting polypeptide was verystable (half-life of >20 hours). When X was tyr, ile, glu, or gln, theresulting polypeptide possessed moderate protein stability (half-life of10-30 minutes). In contrast, the residues arg, lys phe, leu, trp, his,asp, and asn, all conferred low stability on the beta-galactosidasepolypeptide (half-life of <3 minutes). The residue arginine (arg), whenlocated at the amino terminus of a polypeptide, appears to generallyconfer the lowest stability. Thus, chimeric constructs and correspondingchimeric polypeptides employing an arg residue at the position X,described above, are generally preferred embodiments of the presentinvention. This is because a general goal of the invention is toeliminate the function of the target gene polypeptide in the cell.

[0077] The above described experiments establishing the relativehalf-lives conferred by each of the 20 possible amino terminal residuesform the basis of the N-end rule. The N-end rule system components arethose gene products which act to bring about the rapid proteolysis ofpolypeptides possessing amino-terminal residues which conferinstability. The N-end rule system for proteolysis in eucaryotes appearsto be a part of the general ubiquitin-dependent proteolytic systempathways possessed by apparently all eucaryotic cells. Briefly, thissystem involves the covalent tagging of a target polypeptide on one ormore lysine residues by a ubiquitin polypeptide marker (to form a target(lys)-epsilon amino-gly(76) Ubiquitin covalent bond). Additionalubiquitin moieties may be subsequently conjugated to the targetpolypeptide and the resulting “ubiquitinated” target polypeptide is thensubject to complete proteolytic destruction by a large (26S)multiprotein complex known as the proteasome. The enzymes whichconjugate the ubiquitin moieties to the targeted protein include E2 andE3 (or ubiquitin ligase) functions. The E2 and E3 enzymes are thought topossess most of the specificity for ubiquitin dependent proteolyticprocesses.

[0078] Indeed a key component of the N-end rule proteolytic pathway inyeast is UBR1 (Bartel, et al. (1990) EMBO J. 9: 3179-89), a gene whichencodes an E3 like function which appears to recognize polypeptidespossessing susceptible amino terminal residues and thereby facilitatesubiquitination of such polypeptides (Dohmen et al. (1991) Proc. Natl.Acad. Sci. USA 88: 7351-55). In preferred embodiments of the invention,UBR1 is used as the N-end rule component which is the effector ofproteolytic degradation of the target gene polypeptide. The UBR1 genehas now been cloned from a mammalian organism (Kwon et al. (1998) Proc.Natl. Acad. Sci. USA 95: 7893-903) as well as from yeast. Thus theconstruction of a UBR1 mouse knockout is imminent and so both prongs ofthe two-pronged gene function shut-off system can now be set up in bothyeast and mammalian host cells.

[0079] A preferred embodiment of the N-end rule component of thetwo-component shut-off is the above described N-end rule ubiquitinligase UBR1 gene. This gene is particularly convenient since it can beused in conjunction with any of the above described “X” amino-terminaldestabilizing residues including: the most destabilizing—arg; stronglydestabilizing residues—such as lys phe, leu, trp, his, asp, and asn; andmoderately destabilizing residues—such as tyr, ile, glu, or gln. Indeed,it is an object of the present invention to provide a means, wheredesired, to not completely shut-off a target gene's function, but merelyto attenuate it to some set degree. This can be achieved using themethod of the present invention in any of a number of ways. For example,a moderately destabilizing amino-terminal residue (X=tyr, ile, glu, orgin) can be deployed on the target polypeptide—resulting in a less rapidremoval of the target polypeptide pool. Alternatively, only one of thetwo prongs of the method could be employed such as only thetranscriptional repression prong or only the targeted proteolysis prong.

[0080] Alternative embodiments of the N-end rule component of thepresent invention include S. cerevisiae UBC2 (RAD6), which encodes an E2ubiquitin conjugating function which cooperates with the UBR1-encodedN-end rule E3 to promote multiubiquitination and subsequent degradationof N-end rule substrates (Dohmen et al. (1991) Proc. Natl. Acad. Sci.USA 88: 7351-55). Thus N-end rule directed proteolysis will not occur inthe absence of either UBR1 or UBC2. This allows either gene to be usedas the inducible “effector of targeted proteolysis” by the method of thepresent invention. Indeed, a target gene polypeptide possessing an N-endrule destabilizing amino-terminal amino acid (such as arg) will bestable until expression of either the UBR1 (E3) or the UBC2 (E2) isinduced from the cognate inducible promoter construct.

[0081] Both UBR1 and UBC2 can be used in conjunction with any of theabove described “X” amino-terminal destabilizing residues including: themost destabilizing—arg; strongly destabilizing residues—such as lys phe,leu, trp, his, asp, and asn; and moderately destabilizing residues—suchas tyr, ile, glu, or gin. Still other alternative embodiments of theN-end rule component of the present invention are components of theN-end rule system which affect only a subset of the destabilizingresidues. For example, the NTA1 deamidase (Baker and Varshavsky (1995) JBiol Chem 270: 12065-74) functions to deaminate amino-terminal asn orgln residues (to form polypeptides with asp or glu amino-terminalresidues respectively). Yeast strains harboring nta1 null alleles areunable to degrade N-end rule substrates that bear amino-terminal asn orgin residues. Thus, the NTA1 gene is an alternative embodiment of theN-end rule component of the present invention, but is used preferably inconjunction with a target gene polypeptide (X-target), in which X iseither asn or gin. Similarly the ATE1 transferase (Balzi et al. (1990)J. Biol Chem 265: 7464-71) is an enzyme which acts to transfer the argmoiety from a tRNA˜Arg activated tRNA to amino-terminal glu or aspbearing polypeptides. The resulting arg-glu-polypeptide andarg-asp-polypeptide products are then susceptible to the E2/E3-mediatedN-end rule dependent proteolytic processes described above. Thus, theATE1 transferase is an alternative embodiment of the N-end rulecomponent of the present invention, but its use is preferably tied totarget gene polypeptides (X-target), in which X is asp, glu, asn or gln.Polypeptides bearing the latter two amino-terminal residues are firstconverted to polypeptides bearing one of the former tow amino-terminalresidues by NTA1 deamidase function described above.

[0082] It is important to note here that, as is the case for therepressor of the present invention which is made subject to induction byan inducible promoter of the present invention, the N-end rule componentmust be available as a clone so that it can be put under the control ofan inducible promoter (using standard subcloning methods known in theart). This can be achieved by first introducing a genetically engineeredcopies of the inducible repressor and the inducible N-end rule componentconstructs, and subsequently deleting the normal chromosomal copies ofthese genes from the host by “knockout” methods. Such methods, we notehere are well developed in the art—particularly in the case of both theyeast Saccharomyces cerevisiae and the mammal mouse. More convenient,however, is the availability of “knock-in” technology which allows theexisting chromosomal copy of the gene to be modified to so that itsnative promoter is deleted and an inducible promoter is inserted in asingle step. FIG. 2A diagrams this process for the replacement of thenative promoter of the target gene with a repressible promoter, but thisprinciple is also applicable to the replacement of the native promoterof the effector of suppression (i.e. the transcriptional repressorand/or the N-end rule component) with a suitable inducible promoter.

[0083] 4.3.4 Ubiquitin Polypeptide Sequences

[0084] As shown in FIG. 1B, the target gene must be fused downstream ofa codon which encodes an N-end rule susceptible residue (X, as describedabove) and this residue, in term, must be fused in-frame to thecarboxy-terminus of a ubiquitin coding sequence (generally gly76 ofubiquitin). The reason for constructing this extensive chimeric geneconstruct is to take advantage of the ability of constitutive ubiquitinproteases to cleave any peptide bond which is carboxy-terminal to gly76of a ubiquitin moiety. This isopeptidase normally functions to processpoly-ubiquitin chains (the translational product of the tandem ubiquitinencoding sequences of eucaryotic genomes) into discrete (normally 76 aa)ubiquitin moieties which are used in ubiquitin-system pathways. In themethod of the present invention, the ubiquitin isopeptidases serve as aconvenient means to generate target gene polypeptides bearing specificamino-terminal residues (X). Nonetheless, it is understood that otheralternatives to mammalian or yeast ubiquitin exist which can function inthe method of the present invention. Such ubiquitin equivalents include,for example, ubiquitin mutants, ubiquitin-like proteins,ubiquitin-related proteins, and ubiquitin-homologous proteins. Forexample, ubiquitin-like proteins such as NEDD8, UBL1, FUBI, and UCRP, aswell as analogous ubiquitin-related proteins such as SUMO/Sentrin/Pic1may be used as ubiquitin equivalents in the method of the invention.Other proteins related to ubiqutin, but which are somewhat lesshomologous to it, include ubiquitin-homologous proteins such as Rad23and Dsk2 whose similarity to ubiquitin does not include the presence ofa carboxyl-terminal pair of glycines. These ubiquitin-like proteinsshare the common features of being related to ubiquitin by amino acidsequence homology and, with the apparent exception of the ubiquitinhomologous proteins, of being covalently transferred to cellular proteintargets post-translationally.

[0085] Indeed, the intended scope of the immediate invention encompassesany means known in the art by which a target polypeptide bearing anN-end rule susceptible residue (X=arg, lys, his, leu, phe, try, ile,trp, asn, gln, asp, or glu) can be generated.

[0086] 4.3.5 Target Genes

[0087] As discussed above, the method of the present invention isideally suited to the analysis and exploitation of genes whose functionis essential for viability. Moreover, the methods developed here allowvirtually any gene to be made subject to either or both of the genefunction shut-off prongs of the present invention. FIG. 2A diagrams themanner in which virtually any desired target gene comprising a nativepromoter (napr) which drives expression of the target ORF (open readingframe) can be put under the control of a repressible promoter (repr)which is subject to the transcriptional repression prong of the presentinvention. Furthermore, FIG. 2B diagrams the manner in which virtuallyany desired target gene target ORF can be fused in-frame with aUbiquitin-X-Ep (in which Ep is an optional epitope tag which can be usedto facilitate the measurement of target gene polypeptide levels) tocreate a gene sequence which encodes a target gene polypeptide which issubject to the targeted proteolytic destruction prong of the presentinvention. As noted earlier these construct can be created througheither standard subcloning techniques known in the art, or by means of a“knock-in” construct which has the advantage of yielding the desiredaltered target gene in a single step. A detail description of thesuitable mouse cell technology is provided below. FIG. 3 furtherdiagrams how a single target gene can be made subject to control by boththe transcriptional repression and the targeted proteolytic destructionprong of the invention.

[0088] Specific examples of preferred target genes include variouscomponents of the RNApoIII transcriptional machinery, as described inthe Example sections below. These include TAF60, TAF19, TAF90, TAF130,TFIIB, and TBP (see Moqtader et al. (1996) Nature 383: 188-91).

[0089] 4.3.6 Other Methods

[0090] Methods for obtaining transgenic and knockout non-human animalsare well known in the art. Knock out mice are generated by homologousintegration of a “knock out” construct into a mouse embryonic stem cellchromosome which encodes the gene to be knocked out. In one embodiment,gene targeting, which is a method of using homologous recombination tomodify an animal's genome, can be used to introduce changes intocultured embryonic stem cells. By targeting a Target gene of interest inES cells, these changes can be introduced into the germlines of animalsto generate chimeras. The gene targeting procedure is accomplished byintroducing into tissue culture cells a DNA targeting construct thatincludes a segment homologous to a target Target gene locus, and whichalso includes an intended sequence modification to the Target genomicsequence (e.g., insertion, deletion, point mutation). The treated cellsare then screened for accurate targeting to identify and isolate thosewhich have been properly targeted.

[0091] Gene targeting in embryonic stem cells is in fact a schemecontemplated by the present invention as a means for disrupting a Targetgene function through the use of a targeting transgene constructdesigned to undergo homologous recombination with one or more Targetgenomic sequences. The targeting construct can be arranged so that, uponrecombination with an element of a Target gene, a positive selectionmarker is inserted into (or replaces) coding sequences of the gene. Theinserted sequence functionally disrupts the Target gene, while alsoproviding a positive selection trait. Exemplary Target gene targetingconstructs are described in more detail below.

[0092] Generally, the embryonic stem cells (ES cells) used to producethe knockout animals will be of the same species as the knockout animalto be generated. Thus for example, mouse embryonic stem cells willusually be used for generation of knockout mice.

[0093] Embryonic stem cells are generated and maintained using methodswell known to the skilled artisan such as those described by Doetschmanet al. (1985) J. Embryol. Exp. MoMFGFhol. 87:27-45). Any line of EScells can be used, however, the line chosen is typically selected forthe ability of the cells to integrate into and become part of the germline of a developing embryo so as to create germ line transmission ofthe knockout construct. Thus, any ES cell line that is believed to havethis capability is suitable for use herein. One mouse strain that istypically used for production of ES cells, is the 129J strain. AnotherES cell line is murine cell line D3 (American Type Culture Collection,catalog no. CKL 1934) Still another preferred ES cell line is the WW6cell line (loffe et al. (1995) PNAS 92:7357-7361). The cells arecultured and prepared for knockout construct insertion using methodswell known to the skilled artisan, such as those set forth by Robertsonin: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C. [1987]); by Bradley et al.(1986) Current Topics in Devel. Biol. 20:357-371); and by Hogan et al.(Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. [1986]).

[0094] A knock out construct refers to a uniquely configured fragment ofnucleic acid which is introduced into a stem cell line and allowed torecombine with the genome at the chromosomal locus of the gene ofinterest to be mutated. Thus a given knock out construct is specific fora given gene to be targeted for disruption. Nonetheless, many commonelements exist among these constructs and these elements are well knownin the art. A typical knock out construct contains nucleic acidfragments of not less than about 0.5 kb nor more than about 10.0 kb fromboth the 5′ and the 3′ ends of the genomic locus which encodes the geneto be mutated. These two fragments are separated by an interveningfragment of nucleic acid which encodes a positive selectable marker,such as the neomycin resistance gene (neo^(R)). The resulting nucleicacid fragment, consisting of a nucleic acid from the extreme 5′ end ofthe genomic locus linked to a nucleic acid encoding a positiveselectable marker which is in turn linked to a nucleic acid from theextreme 3′ end of the genomic locus of interest, omits most of thecoding sequence for Target gene or other gene of interest to be knockedout. When the resulting construct recombines homologously with thechromosome at this locus, it results in the loss of the omitted codingsequence, otherwise known as the structural gene, from the genomiclocus. A stem cell in which such a rare homologous recombination eventhas taken place can be selected for by virtue of the stable integrationinto the genome of the nucleic acid of the gene encoding the positiveselectable marker and subsequent selection for cells expressing thismarker gene in the presence of an appropriate drug (neomycin in thisexample).

[0095] Variations on this basic technique also exist and are well knownin the art. For example, a “knock-in” construct refers to the same basicarrangement of a nucleic acid encoding a 5′ genomic locus fragmentlinked to nucleic acid encoding a positive selectable marker which inturn is linked to a nucleic acid encoding a 3′ genomic locus fragment,but which differs in that none of the coding sequence is omitted andthus the 5′ and the 3′ genomic fragments used were initially contiguousbefore being disrupted by the introduction of the nucleic acid encodingthe positive selectable marker gene. This “knock-in” type of constructis thus very useful for the construction of mutant transgenic animalswhen only a limited region of the genomic locus of the gene to bemutated, such as a single exon, is available for cloning and geneticmanipulation. Alternatively, the “knock-in” construct can be used tospecifically eliminate a single functional domain of the targetted gene,resulting in a transgenic animal which expresses a polypeptide of thetargetted gene which is defective in one function, while retaining thefunction of other domains of the encoded polypeptide. This type of“knock-in” mutant frequently has the characteristic of a so-called“dominant negative” mutant because, especially in the case of proteinswhich homomultimerize, it can specifically block the action of (or“poison”) the polypeptide product of the wild-type gene from which itwas derived. In a variation of the knock-in technique, a marker gene isintegrated at the genomic locus of interest such that expression of themarker gene comes under the control of the transcriptional regulatoryelements of the targeted gene. A marker gene is one that encodes anenzyme whose activity can be detected (e.g., b-galactosidase), theenzyme substrate can be added to the cells under suitable conditions,and the enzymatic activity can be analyzed. One skilled in the art willbe familiar with other useful markers and the means for detecting theirpresence in a given cell. All such markers are contemplated as beingincluded within the scope of the teaching of this invention.

[0096] As mentioned above, the homologous recombination of the abovedescribed “knock out” and “knock in” constructs is very rare andfrequently such a construct inserts nonhomologously into a random regionof the genome where it has no effect on the gene which has been targetedfor deletion, and where it can potentially recombine so as to disruptanother gene which was otherwise not intended to be altered. Suchnonhomologous recombination events can be selected against by modifyingthe abovementioned knock out and knock in constructs so that they areflanked by negative selectable markers at either end (particularlythrough the use of two allelic variants of the thymidine kinase gene,the polypeptide product of which can be selected against in expressingcell lines in an appropriate tissue culture medium well known in theart—i.e. one containing a drug such as 5-bromodeoxyuridine). Thus apreferred embodiment of such a knock out or knock in construct of theinvention consist of a nucleic acid encoding a negative selectablemarker linked to a nucleic acid encoding a 5′ end of a genomic locuslinked to a nucleic acid of a positive selectable marker which in turnis linked to a nucleic acid encoding a 3′ end of the same genomic locuswhich in turn is linked to a second nucleic acid encoding a negativeselectable marker Nonhomologous recombination between the resultingknock out construct and the genome will usually result in the stableintegration of one or both of these negative selectable marker genes andhence cells which have undergone nonhomologous recombination can beselected against by growth in the appropriate selective media (e.g.media containing a drug such as 5-bromodeoxyuridine for example).Simultaneous selection for the positive selectable marker and againstthe negative selectable marker will result in a vast enrichment forclones in which the knock out construct has recombined homologously atthe locus of the gene intended to be mutated. The presence of thepredicted chromosomal alteration at the targeted gene locus in theresulting knock out stem cell line can be confirmed by means of Southernblot analytical techniques which are well known to those familiar in theart. Alternatively, PCR can be used.

[0097] Each knockout construct to be inserted into the cell must firstbe in the linear form. Therefore, if the knockout construct has beeninserted into a vector (described infra), linearization is accomplishedby digesting the DNA with a suitable restriction endonuclease selectedto cut only within the vector sequence and not within the knockoutconstruct sequence.

[0098] For insertion, the knockout construct is added to the ES cellsunder appropriate conditions for the insertion method chosen, as isknown to the skilled artisan. For example, if the ES cells are to beelectroporated, the ES cells and knockout construct DNA are exposed toan electric pulse using an electroporation machine and following themanufacturer's guidelines for use. After electroporation, the ES cellsare typically allowed to recover under suitable incubation conditions.The cells are then screened for the presence of the knock out constructas explained above. Where more than one construct is to be introducedinto the ES cell, each knockout construct can be introducedsimultaneously or one at a time.

[0099] After suitable ES cells containing the knockout construct in theproper location have been identified by the selection techniquesoutlined above, the cells can be inserted into an embryo. Insertion maybe accomplished in a variety of ways known to the skilled artisan,however a preferred method is by microinjection. For microinjection,about 10-30 cells are collected into a micropipet and injected intoembryos that are at the proper stage of development to permitintegration of the foreign ES cell containing the knockout constructinto the developing embryo. For instance, the transformed ES cells canbe microinjected into blastocytes. The suitable stage of development forthe embryo used for insertion of ES cells is very species dependent,however for mice it is about 3.5 days. The embryos are obtained byperfusing the uterus of pregnant females. Suitable methods foraccomplishing this are known to the skilled artisan, and are set forthby, e.g., Bradley et al. (supra).

[0100] While any embryo of the right stage of development is suitablefor use, preferred embryos are male. In mice, the preferred embryos alsohave genes coding for a coat color that is different from the coat colorencoded by the ES cell genes. In this way, the offspring can be screenedeasily for the presence of the knockout construct by looking for mosaiccoat color (indicating that the ES cell was incorporated into thedeveloping embryo). Thus, for example, if the ES cell line carries thegenes for white fur, the embryo selected will carry genes for black orbrown fur.

[0101] After the ES cell has been introduced into the embryo, the embryomay be implanted into the uterus of a pseudopregnant foster mother forgestation. While any foster mother may be used, the foster mother istypically selected for her ability to breed and reproduce well, and forher ability to care for the young. Such foster mothers are typicallyprepared by mating with vasectomized males of the same species. Thestage of the pseudopregnant foster mother is important for successfulimplantation, and it is species dependent. For mice, this stage is about2-3 days pseudopregnant.

[0102] Offspring that are born to the foster mother may be screenedinitially for mosaic coat color where the coat color selection strategy(as described above, and in the appended examples) has been employed. Inaddition, or as an alternative, DNA from tail tissue of the offspringmay be screened for the presence of the knockout construct usingSouthern blots and/or PCR as described above. Offspring that appear tobe mosaics may then be crossed to each other, if they are believed tocarry the knockout construct in their germ line, in order to generatehomozygous knockout animals. Homozygotes may be identified by Southernblotting of equivalent amounts of genomic DNA from mice that are theproduct of this cross, as well as mice that are known heterozygotes andwild type mice.

[0103] Other means of identifying and characterizing the knockoutoffspring are available. For example, Northern blots can be used toprobe the mRNA for the presence or absence of transcripts encodingeither the gene knocked out, the marker gene, or both. In addition,Western blots can be used to assess the level of expression of the MFGFgene knocked out in various tissues of the offspring by probing theWestern blot with an antibody against the particular MFGF protein, or anantibody against the marker gene product, where this gene is expressed.Finally, in situ analysis (such as fixing the cells and labeling withantibody) and/or FACS (fluorescence activated cell sorting) analysis ofvarious cells from the offspring can be conducted using suitableantibodies to look for the presence or absence of the knockout constructgene product.

[0104] Yet other methods of making knock-out or disruption transgenicanimals are also generally known. See, for example, Manipulating theMouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1986). Recombinase dependent knockouts can also be generated, e.g.by homologous recombination to insert target sequences, such that tissuespecific and/or temporal control of inactivation of a Target-gene can becontrolled by recombinase sequences (described infra).

[0105] Animals containing more than one knockout construct and/or morethan one transgene expression construct are prepared in any of severalways. The preferred manner of preparation is to generate a series ofmammals, each containing one of the desired transgenic phenotypes. Suchanimals are bred together through a series of crosses, backcrosses andselections, to ultimately generate a single animal containing alldesired knockout constructs and/or expression constructs, where theanimal is otherwise congenic (genetically identical) to the wild typeexcept for the presence of the knockout construct(s) and/ortransgene(s).

[0106] A Target transgene can encode the wild-type form of the protein,or can encode homologs thereof, including both agonists and antagonists,as well as antisense constructs. In preferred embodiments, theexpression of the transgene is restricted to specific subsets of cells,tissues or developmental stages utilizing, for example, cis-actingsequences that control expression in the desired pattern. In the presentinvention, such mosaic expression of a Target gene protein can beessential for many forms of lineage analysis and can additionallyprovide a means to assess the effects of, for example, lack of Targetgene expression which might grossly alter development in small patchesof tissue within an otherwise normal embryo. Toward this and,tissue-specific regulatory sequences and conditional regulatorysequences can be used to control expression of the transgene in certainspatial patterns. Moreover, temporal patterns of expression can beprovided by, for example, conditional recombination systems orprokaryotic transcriptional regulatory sequences.

[0107] Genetic techniques, which allow for the expression of transgenescan be regulated via site-specific genetic manipulation in vivo, areknown to those skilled in the art. For instance, genetic systems areavailable which allow for the regulated expression of a recombinase thatcatalyzes the genetic recombination of a target sequence. As usedherein, the phrase “target sequence” refers to a nucleotide sequencethat is genetically recombined by a recombinase. The target sequence isflanked by recombinase recognition sequences and is generally eitherexcised or inverted in cells expressing recombinase activity.Recombinase catalyzed recombination events can be designed such thatrecombination of the target sequence results in either the activation orrepression of expression of one of the subject Target gene proteins. Forexample, excision of a target sequence which interferes with theexpression of a recombinant Target gene, such as one which encodes anantagonistic homolog or an antisense transcript, can be designed toactivate expression of that gene. This interference with expression ofthe protein can result from a variety of mechanisms, such as spatialseparation of the Target gene from the promoter element or an internalstop codon. Moreover, the transgene can be made wherein the codingsequence of the gene is flanked by recombinase recognition sequences andis initially transfected into cells in a 3′ to 5′ orientation withrespect to the promoter element. In such an instance, inversion of thetarget sequence will reorient the subject gene by placing the 5′ end ofthe coding sequence in an orientation with respect to the promoterelement which allow for promoter driven transcriptional activation.

[0108] The transgenic animals of the present invention all includewithin a plurality of their cells a transgene of the present invention,which transgene alters the phenotype of the “host cell” with respect toregulation of cell growth, death and/or differentiation. Since it ispossible to produce transgenic organisms of the invention utilizing oneor more of the transgene constructs described herein, a generaldescription will be given of the production of transgenic organisms byreferring generally to exogenous genetic material. This generaldescription can be adapted by those skilled in the art in order toincorporate specific transgene sequences into organisms utilizing themethods and materials described below.

[0109] In an illustrative embodiment, either the cre/loxP recombinasesystem of bacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orbanet al. (1992) PNAS 89:6861-6865) or the FLP recombinase system ofSaccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355;PCT publication WO 92/15694) can be used to generate in vivosite-specific genetic recombination systems. Cre recombinase catalyzesthe site-specific recombination of an intervening target sequencelocated between loxP sequences. loxP sequences are 34 base pairnucleotide repeat sequences to which the Cre recombinase binds and arerequired for Cre recombinase mediated genetic recombination. Theorientation of loxP sequences determines whether the intervening targetsequence is excised or inverted when Cre recombinase is present(Abremski et al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing theexcision of the target sequence when the loxP sequences are oriented asdirect repeats and catalyzes inversion of the target sequence when loxPsequences are oriented as inverted repeats.

[0110] Accordingly, genetic recombination of the target sequence isdependent on expression of the Cre recombinase. Expression of therecombinase can be regulated by promoter elements which are subject toregulatory control, e.g., tissue-specific, developmental stage-specific,inducible or repressible by externally added agents. This regulatedcontrol will result in genetic recombination of the target sequence onlyin cells where recombinase expression is mediated by the promoterelement. Thus, the activation expression of a recombinant Target geneprotein can be regulated via control of recombinase expression.

[0111] Use of the cre/loxP recombinase system to regulate expression ofa recombinant Target gene protein requires the construction of atransgenic animal containing transgenes encoding both the Crerecombinase and the subject protein. Animals containing both the Crerecombinase and a recombinant Target gene can be provided through theconstruction of “double” transgenic animals. A convenient method forproviding such animals is to mate two transgenic animals each containinga transgene, e.g., a Target gene and recombinase gene.

[0112] One advantage derived from initially constructing transgenicanimals containing a Target transgene in a recombinase-mediatedexpressible format derives from the likelihood that the subject protein,whether agonistic or antagonistic, can be deleterious upon expression inthe transgenic animal. In such an instance, a founder population, inwhich the subject transgene is silent in all tissues, can be propagatedand maintained. Individuals of this founder population can be crossedwith animals expressing the recombinase in, for example, one or moretissues and/or a desired temporal pattern. Thus, the creation of afounder population in which, for example, an antagonistic Targettransgene is silent will allow the study of progeny from that founder inwhich disruption of Target gene mediated induction in a particulartissue or at certain developmental stages would result in, for example,a lethal phenotype.

[0113] Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the Target transgene.Exemplary promoters and the corresponding trans-activating prokaryoticproteins are given in U.S. Pat. No. 4,833,080.

[0114] Moreover, expression of the conditional transgenes can be inducedby gene therapy-like methods wherein a gene encoding thetrans-activating protein, e.g. a recombinase or a prokaryotic protein,is delivered to the tissue and caused to be expressed, such as in acell-type specific manner. By this method, a Target A transgene couldremain silent into adulthood until “turned on” by the introduction ofthe trans-activator.

[0115] In an exemplary embodiment, the “transgenic non-human animals” ofthe invention are produced by introducing transgenes into the germlineof the non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thespecific line(s) of any animal used to practice this invention areselected for general good health, good embryo yields, good pronuclearvisibility in the embryo, and good reproductive fitness. In addition,the haplotype is a significant factor. For example, when transgenic miceare to be produced, strains such as C57BL/6 or FVB lines are often used(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those withH-2^(b), H-2^(d) or H-2^(q) haplotypes such as C57BL/6 or DBA/1. Theline(s) used to practice this invention may themselves be transgenics,and/or may be knockouts (i.e., obtained from animals which have one ormore genes partially or completely suppressed).

[0116] In one embodiment, the transgene construct is introduced into asingle stage embryo. The zygote is the best target for micro-injection.In the mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1-2 pl ofDNA solution. The use of zygotes as a target for gene transfer has amajor advantage in that in most cases the injected DNA will beincorporated into the host gene before the first cleavage (Brinster etal. (1985) PNAS 82:4438-4442). As a consequence, all cells of thetransgenic animal will carry the incorporated transgene. This will ingeneral also be reflected in the efficient transmission of the transgeneto offspring of the founder since 50% of the germ cells will harbor thetransgene.

[0117] Normally, fertilized embryos are incubated in suitable mediauntil the pronuclei appear. At about this time, the nucleotide sequencecomprising the transgene is introduced into the female or malepronucleus as described below. In some species such as mice, the malepronucleus is preferred. It is most preferred that the exogenous geneticmaterial be added to the male DNA complement of the zygote prior to itsbeing processed by the ovum nucleus or the zygote female pronucleus. Itis thought that the ovum nucleus or female pronucleus release moleculeswhich affect the male DNA complement, perhaps by replacing theprotamines of the male DNA with histones, thereby facilitating thecombination of the female and male DNA complements to form the diploidzygote.

[0118] Thus, it is preferred that the exogenous genetic material beadded to the male complement of DNA or any other complement of DNA priorto its being affected by the female pronucleus. For example, theexogenous genetic material is added to the early male pronucleus, assoon as possible after the formation of the male pronucleus, which iswhen the male and female pronuclei are well separated and both arelocated close to the cell membrane. Alternatively, the exogenous geneticmaterial could be added to the nucleus of the sperm after it has beeninduced to undergo decondensation. Sperm containing the exogenousgenetic material can then be added to the ovum or the decondensed spermcould be added to the ovum with the transgene constructs being added assoon as possible thereafter.

[0119] Introduction of the transgene nucleotide sequence into the embryomay be accomplished by any means known in the art such as, for example,microinjection, electroporation, or lipofection. Following introductionof the transgene nucleotide sequence into the embryo, the embryo may beincubated in vitro for varying amounts of time, or reimplanted into thesurrogate host, or both. In vitro incubation to maturity is within thescope of this invention. One common method in to incubate the embryos invitro for about 1-7 days, depending on the species, and then reimplantthem into the surrogate host.

[0120] For the purposes of this invention a zygote is essentially theformation of a diploid cell which is capable of developing into acomplete organism. Generally, the zygote will be comprised of an eggcontaining a nucleus formed, either naturally or artificially, by thefusion of two haploid nuclei from a gamete or gametes. Thus, the gametenuclei must be ones which are naturally compatible, i.e., ones whichresult in a viable zygote capable of undergoing differentiation anddeveloping into a functioning organism. Generally, a euploid zygote ispreferred. If an aneuploid zygote is obtained, then the number ofchromosomes should not vary by more than one with respect to the euploidnumber of the organism from which either gamete originated.

[0121] In addition to similar biological considerations, physical onesalso govern the amount (e.g., volume) of exogenous genetic materialwhich can be added to the nucleus of the zygote or to the geneticmaterial which forms a part of the zygote nucleus. If no geneticmaterial is removed, then the amount of exogenous genetic material whichcan be added is limited by the amount which will be absorbed withoutbeing physically disruptive. Generally, the volume of exogenous geneticmaterial inserted will not exceed about 10 picoliters. The physicaleffects of addition must not be so great as to physically destroy theviability of the zygote. The biological limit of the number and varietyof DNA sequences will vary depending upon the particular zygote andfunctions of the exogenous genetic material and will be readily apparentto one skilled in the art, because the genetic material, including theexogenous genetic material, of the resulting zygote must be biologicallycapable of initiating and maintaining the differentiation anddevelopment of the zygote into a functional organism.

[0122] The number of copies of the transgene constructs which are addedto the zygote is dependent upon the total amount of exogenous geneticmaterial added and will be the amount which enables the genetictransformation to occur. Theoretically only one copy is required;however, generally, numerous copies are utilized, for example,1,000-20,000 copies of the transgene construct, in order to insure thatone copy is functional. As regards the present invention, there willoften be an advantage to having more than one functioning copy of eachof the inserted exogenous DNA sequences to enhance the phenotypicexpression of the exogenous DNA sequences.

[0123] Any technique which allows for the addition of the exogenousgenetic material into nucleic genetic material can be utilized so longas it is not destructive to the cell, nuclear membrane or other existingcellular or genetic structures. The exogenous genetic material ispreferentially inserted into the nucleic genetic material bymicroinjection. Microinjection of cells and cellular structures is knownand is used in the art.

[0124] Reimplantation is accomplished using standard methods. Usually,the surrogate host is anesthetized, and the embryos are inserted intothe oviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

[0125] Transgenic offspring of the surrogate host may be screened forthe presence and/or expression of the transgene by any suitable method.Screening is often accomplished by Southern blot or Northern blotanalysis, using a probe that is complementary to at least a portion ofthe transgene. Western blot analysis using an antibody against theprotein encoded by the transgene may be employed as an alternative oradditional method for screening for the presence of the transgeneproduct. Typically, DNA is prepared from tail tissue and analyzed bySouthern analysis or PCR for the transgene. Alternatively, the tissuesor cells believed to express the transgene at the highest levels aretested for the presence and expression of the transgene using Southernanalysis or PCR, although any tissues or cell types may be used for thisanalysis.

[0126] Alternative or additional methods for evaluating the presence ofthe transgene include, without limitation, suitable biochemical assayssuch as enzyme and/or immunological assays, histological stains forparticular marker or enzyme activities, flow cytometric analysis, andthe like. Analysis of the blood may also be useful to detect thepresence of the transgene product in the blood, as well as to evaluatethe effect of the transgene on the levels of various types of bloodcells and other blood constituents.

[0127] Progeny of the transgenic animals may be obtained by mating thetransgenic animal with a suitable partner, or by in vitro fertilizationof eggs and/or sperm obtained from the transgenic animal. Where matingwith a partner is to be performed, the partner may or may not betransgenic and/or a knockout; where it is transgenic, it may contain thesame or a different transgene, or both. Alternatively, the partner maybe a parental line. Where in vitro fertilization is used, the fertilizedembryo may be implanted into a surrogate host or incubated in vitro, orboth. Using either method, the progeny may be evaluated for the presenceof the transgene using methods described above, or other appropriatemethods.

[0128] The transgenic animals produced in accordance with the presentinvention will include exogenous genetic material. As set out above, theexogenous genetic material will, in certain embodiments, be a DNAsequence which results in the production of a Target protein (eitheragonistic or antagonistic), and antisense transcript, or a Targetmutant. Further, in such embodiments the sequence will be attached to atranscriptional control element, e.g., a promoter, which preferablyallows the expression of the transgene product in a specific type ofcell.

[0129] Retroviral infection can also be used to introduce transgene intoa non-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

[0130] A third type of target cell for transgene introduction is theembryonal stem cell (ES). ES cells are obtained from pre-implantationembryos cultured in vitro and fused with embryos (Evans et al. (1981)Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler etal. (1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

5. EXAMPLES

[0131] The present invention is further illustrated by the followingexamples which should not be construed as limiting in any way. Thecontents of all cited references (including literature references,issued patents, published patent applications as cited throughout thisapplication are hereby expressly incorporated by reference.

[0132] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, microbiology and recombinant DNA, which arewithin the skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.),Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987).

5.1 Example 1

[0133] Use of Double Shutoff Yeast Strain Containing Copper-InducibleYeast Alleles of Both ROX1 and UBR1 to Determine that TBP-AssociatedFactors are not Generally Required for Transcriptional Activation inYeast

[0134] Materials and Methods

[0135] The parent strain ZMY60, containing copper-inducible alleles ofUBR1 and ROX1, was created as follows. A cassette containing thecopper-inducible derivative of the H1S3 promoter (Klein, C and K. Struhl(1994) Science 266: pp. 280-282) and 2 kb of upstream flanking sequencewas inserted at the initial ATG of a plasmid-borne genomic fragment ofROX1 to create the URA3 integrating plasmid ZM195. The same cassette wasinserted at the initial ATG of UBR1 to create ZM197. Both of thesecopper-driven alleles were introduced into yeast strain KY114 (Iyer, Vand K. Struhl (1995) Mol. Cell. Biol. 116: pp. 7069-7086) in successivetwo-step gene replacements. To create TAF disruption molecules, anothercassette comprising an inframe fusion of ubiquitin, arginine, LacI andthe HA epitope-driven by the ANB1 promoter, was fused in-frame to ashort 5′ fragment of TAF coding sequence beginning at the initial TAFATG. To create a given conditional knockout strain, the relevant TAFknockout molecule on a URA3 integrating plasmid was linearized withinthe TAF coding sequence fragment and transformed into ZMY60. Thisintegration results in homologous recombination at the TAF locus toyield a short, non-functional 5′ TAF piece under its normal promoter,followed immediately downstream by a full-length copy of the tagged TAFunder the ANB1 promoter. Selection for uracil prototrophy was maintainedin all experiments to avoid loss of the integrated plasmid.

[0136] RNA levels were determined by the quantitative S1 analysis asdescribed (Cormack, B. P. et. al., (1994) Genes Dev. 8: pp. 1335-1343;Iyer, V., and K. Struhl (1996) Proc. Natl. Acad. Sci. USA 98: pp.5208-5212). All hybridization reactions contained at least two probes,such that the relative levels of all transcripts were internallycontrolled. The error for any particular RNA determination is ±30%. TAFlevels were determined by western blotting, with bands being detected bychemiluminescence (ECL). Relative levels of TAFs at various times weredetermined by comparing band intensities to serially diluted samplesfrom wild-type cells.

[0137] Results

[0138] As described above, a two-pronged approach was used to createstrains with conditional TAF alleles in which the addition of copper ionleads to the simultaneous cessation of TAF messenger RNA synthesis anddestruction of any TAF protein present in the cell. Strains withconditional alleles of TAF130 (TAF145), TAF90, TAF60 and TAF19 (Fun81),which are homologous to human TAF250, Drosophila TAF80, Drosophila TAF60and human TAF18, respectively, were generated. Of these, TAF130 isparticularly interesting, as it appears to be the scaffold on which theremaining TAFs assemble into the TFIID complex (Chen, J.-L., et. al.,(1994) Cell 79: pp. 93-105) As controls, strains containing conditionalalleles of TFIIB and TBP were generated. In all cases, the conditionalknockout strains fail to grow on copper-containing medium, and theaddition of copper ion caused cells to stop growing within about sixhours. TAF90-depleted cells arrest frequently as large, budding cells,whereas cells depleted of other TAFs display variable and abnormalmorphologies.

[0139] In general, transcription was analysed 8 hours after copper ionaddition, when more than 95% of the cells were dead (they were unable togrow when returned to medium lacking copper). At this time, westernblotting reveals that levels of TAF130, TAF90 and TBP are less than 5%of wild type. Although TAFs may not be completely eliminated by thisprocedure, they are reduced to less than 100-200 molecules per cell(Walker, S. S., et. al., (1996) Nature 385 pp. 185-188), which isconsiderably less than the number of Pol II promoters per cell (-6,000).

[0140] When cells are grown under standard conditions, TAF depletionaffects transcription of selected Pol II promoters. Depletion of TAF130,TAF60, TAF90 and TAF 19 does not significantly affect transcription ofded1 or his3+13, promoters with canonical TATA elements (Chen, W. and K.Struhl (1985) EMBO J, 4: pp. 3272-3280; Iyer, V. and K. Struhl (1995)Mol. Cell. Biol., 15: pp. 7059-7066). However, depletion of TAF130significantly reduces the level of the trp3 and his3+1 transcripts,which arise from promoters with suboptimal, nonconsensus TATA elements(Iyer, V. and K. Struhl (1995) Mol. Cell. Biol. 15: pp. 7059-7066;Martens, J. A. and Brandt, C. J. (1994) J. Biol. Chem., 269: pp.15661-15667). This preferential effect on transcription from promoterscontaining weak TATA elements is also observed when TAF 19 is depleted,albeit to a lesser extent and with slower kinetics, but it does notoccur upon depletion of TAF90 or TAF60. Interestingly, thetranscriptional pattern resulting from TAF130 or TAF19 depletion issimilar to that mediated in yeast by human TBP, which has been suggestedto interact inefficiently with yeast TAFs (Cormack, B. P., et. al.(1994) Genes. Dev. 8: pp. 1335-1343). As expected, depletion of TBP orTFIIB results in a rapid and large reduction of all mRNA species tested.At a late time point (11 hours), depletion of TAF90 confers a moderatedecrease of all transcripts. It is unclear whether this effect reflectsa specific function of TAF90 or arises indirectly from cell death.

[0141] Surprisingly, when the conditional knockout strains are grownunder conditions that support activation by Gcn4 or Ace1, TAF depletiondoes not significantly affect the level of activated transcription(Ace1-dependent activation appears slightly reduced upon TAF90depletion). In contrast, depletion of TFIIB causes the loss of activatedtranscription in both situations. The observed Gcn4- and Ace1-activatedtranscription reflects initiation events that occur under conditions ofTAF depletion, because mRNA half-lives are very short in comparison tothe timescale of the experiment.

[0142] One explanation for the maintenance of Gcn4 and Ace1 activationafter TAF depletion is that TAFs present in active transcriptioncomplexes might be preferentially sequestered from Ubr 1-dependentdegradation. To examine whether active transcription complexes could beassembled after TAF depletion, the conditional knockout strains weregrown in non-inducing conditions, treated with copper for 8 hours, andthen tested for the ability to mediate activator-dependent transcriptionde novo. All the TAF-depleted strains show significant activation byGal4 and heat-shock factor upon exposure to the relevant inducer,whereas activation is not observed in the TFIIB-depleted strain.Similarly, efficient Ace1-dependent activation was observed after TAFswere depleted either by placing the TAF genes under the control of theGAL1, 10 promoter and shifting cells to glucose or by shifting a tsm1(dTAF150 homologue) strain to the restrictive temperature. Theheat-shock and Ace1 activation responses in the TAF-depleted strains arecomparable to the parental strain; Gal4 activation is reduced 3-4 fold.However, as very small fluctuations in Gal4 levels or changes in growthpotential can have pronounced effects on Gal4-dependent transcription(Griggs, D. W. and M. Johnston (1991) Proc. Natl. Acad. Sci. USA, 88 pp.8597-8601), it is unclear whether the decrease reflects a mildactivation defect or whether Gal4 expression is slightly perturbed forother reasons. Previously described activation-defective yeast strainsare considerably more impaired for Gal4-dependent activation, and theyare defective in the response to other acidic activators (Arndt, K. M.et. al., (1995) EMBO J, 14: pp. 1490-1497; Lee, M. and K. Struhl (1995)Mol. Cell. Biol. 15: 5461-5469; Stargell, L. A. and Struhl, K. (1995)Science, 269: pp. 75-78).

[0143] Since TAF depletion does not significantly affect activation byGcn4, Ace1, Gal4, Hsf, and unidentified activators involved in ded1 andhis3+13 transcription, TAFs do not appear to be required fortranscriptional activation in yeast cells. This conclusion was reachedindependently in experiments where TAF depletion was obtained usingtemperature-sensitive mutants or a glucose shutoff procedure (Hernandez,N. (1993) Genes Dev., 7: pp. 1291-1308). It is particularly strikingthat this is true of TAF130, which provides the scaffold for TAFassembly and without which TFIID is likely to be disrupted. AlthoughTAFs are not generally required for transcriptional activation, they areessential for cell growth. One possibility is that TAFs are required forthe response to a subset of activators that affect one or more essentialgenes. Alternatively, TAFs could subtly affect activation of many genes,such that the cumulative effects lead to cell inviability. Finally, assuggested by the effects on trp3 and his3+1 transcription, TAFs may beimportant for transcription from promoters with weak TATA elements.

[0144] This conclusion is in apparent contrast to numerous experimentsin vitro, which indicate that TAFs are crucial in all activatedtranscription. This probably does not indicate that yeast TAFs are lessimportant than their mammalian and Drosophila counterparts because: (a)TAFs are strongly conserved among eukaryotes; (2) TAF-dependentactivation in vitro can be achieved with yeast components (Reese, J. et.al. (1994) Nature, 371: 523-527; Poon, D. et. al. (1995) Proc. Natl.Acad. Sci USA, 92: 8224-8228); and (3) activation can occur in a hamstercell line in which TAF250 (yeast TAF130 homologue) has been thermallyinactivated (Fos transcription occurs normally, and it is unclearwhether the reduction of cyclin A transcription is an indirect effect ofcell-cycle arrest or a direct effect of TAF250) (Wang, E. H. and Tijian,R., (1994) Science, 263: pp. 811-814). A more likely explanation is thatTAFs are functionally redundant with other factors that are absent intypical in vitro reactions. Indeed, activated transcription in theapparent absence of TAFs can occur in vitro when reactions eithercontain Pol II holoenzyme (Koleske, A. J. and Young, R. A. (1994)Nature, 368: pp. 446-469; Kim, Y.-J. et. al., (1994), Cell, 77: 599-608)or are performed on chromatin templates (Balasubramanian, B., et. al.,(1993) Mol. Cell. Biol. 13: pp. 6071-6078). Moreover, most in vitrotranscription reactions are reconstituted with core Pol II, and hencemay lack components of the Pol II holoenzyme (for example, Srb proteins,Gal 11) that are functionally important in vivo (Koleske, A. J. andYoung, R. A. (1995) Trends Biochem. Sci., 20: pp. 113-116).

[0145] A common view of the transcriptional activation process is thatactivator proteins stabilize the Pol II machinery at the promoter,thereby permitting increased transcriptional initiation (Struhl, K.(1996) Cell, 84: pp. 179-182). In principle, activator proteins caninteract with individual components of the Pol II machinery, and indeed,artificial connection of enhancer-bound proteins to TBP (Chatterjee, S.and Struhl, K., (1995) Nature, 374: pp. 820-822; Klages, N. and Strubin,M., (1995), Nature, 374: 822-823), TAFs and components of the Pol IIholoenzyne (Barberis, A. et. al. (1995) Cell, 81: 359-368) can bypassthe need for an activation domain. If natural activators interact withmultiple components, individual components such as TAFs are likely to benon-essential for activation, even if they are potential targets. Thus,although it is possible to generate conditions in which TAFs arerequired for activation in vitro, they do not appear to be generallyrequired in vivo. However, at promoters lacking conventional TATAelements, which are inherently weak targets for TFIID, interactions ofTAFs with basic transcription factors or with promoter DNA may beimportant for stabilizing the Pol II machinery.

5.2 Example 2

[0146] The following example demonstrates how any given target gene canbe configured for the double shutoff system in saccharomyces cervisiae.The system requires two basic components: first, a parent straincontaining copper-inducible alleles of both ROX1 and UBR1; and second, ashort 5′ fragment of the target gene of interest fused in frame to aubiquitin-arginine-lacI-HA (“URLF”) cassette and driven by the ANB1promoter.

[0147] The strain ZMY60 has the genotype: MAT ay, ACE-UBR1, Ace-ROX1,trp1-D1, ura3-52, LEU2, HIS3, ade2-101 in a KY114 background. Togenerate a parent strain in a different background, one utilizes theURA3 integrating plasmids ZM195 and AM197, which must be used insuccessive two-step gene replacements to generate a parent straincontaining copper-inducible ROX1 and UBR1. To create double-shutoffparent strain, one integrates ZM195 into a desired strain with AflII.The URA3 marker is the loopout on FOA (5-fluorotica acid). One wouldthen check for correct loopouts by Southern blotting analysis. Next, onetransforms a correctly ROX1-replaced strain with AatII-digested AM197and one loopout on FOA. Check by Southern.

[0148] {Southern details: With the ZM195 integration, digest with PvuII,and probe with a 5′ piece of the ROX1 ORF (for example, the 550 bpcla1-Pst1 fragment of ZM195). Correct loopouts will pick up a band atabout 3 kb, wildtype ROX1 at about 1 kb.

[0149] With ZM197, digest with Stu1 and Bgl2, and probe with a 5′ pieceof the UBR1 ORF (e.g., the 550 bp Hind3 fragment of ZM197). Correctloopouts will again be 2 kb larger than incorrect ones.}

[0150] To create the ANB-URLF gene fusion, digest or PCR out a short,nonfunctional (usually about 300 bp) fragment the gene beginning at theinitial ATG. The fragment should contain a convenient unique restrictionsite (not too close to either end, and preferably closer to the 3′ endof your fragment than to the fusion junction) to allow for efficientintegration of the final plasmid at the normal gene locus. Using thereading frame information provided, clone the fragment in frame intoZM168, and then transfer the resulting ANB-URLF-gene fragment into adesired yeast integrating vector. Alternatively, both steps can be doneat once with a 3-piece ligation.

[0151] To use the system:

[0152] 1. Digest the ANB-URLF-gene construct with the internalrestriction enzyme, and transform the resulting product into ZMY60 orthe parent strain. Plate the transformants onto synthetic completeplates lacking the plasmid marker.

[0153] 2. Test the transformants on plates lacking or containing 500 μMcopper sulfate. Be sure to maintain selection for the marker so as notto allow looping out of the integrated plasmid. If the gene isessential, the cells should fail to grow in the presence of copper. Moreor less copper may be used depending on the level of shutoff seen withthe particular gene, but in general, 500 μM is effective.

5.3 Example 3

[0154] DKO strains and constructs of TAF 19 (strain ZMY67), TAF60(ZMY66), TAF90 (ZMY68), TAF130 (ZMY69, TBP, AND TFIIB (ZMY71).

[0155] Strain ZMY60 (the DKO parent strain)

[0156] Useful saccharomyces cervisiae strains:

[0157] Parent strains:

[0158] ZMY59 (Ace-UBR1 only strain)

[0159] ZMY61 (Ace-ROX1 only strain)

[0160] ZMY103 (ZMY60 his3Δ200)

[0161] ZMY117 (ZMY60 leu2::PET56)

[0162] ZMY118 (ZMY60 his3Δ200, leu2::PET56)

[0163] shutoff strains:

[0164] ZMY65 (TSM1 DKO)

[0165] ZMY70 (HIS3 DKO))

[0166] ZMY75 (triple-HA-tagged TAF 130 DKO)

[0167] ZMY76 (has complete gene replacement of TAF 130 for ANB-URLF-TAF130)

[0168] ZMY119 (TOA1 DKO strain)

[0169] ZMY95 (triple-HA-tagged TAF23 DKO)

[0170] ZMY96 (triple-HA-tagged TAF40 DKO)

[0171] ZMY97 (triple-HA-tagged TAF67 KO)

[0172] ZMY131 (TAF17 DKO)

[0173] ZMY133 (triple-HA-tagged TAF60 KO)

[0174] ZMY134 (TAF60 DKO-complete gene replacement)

[0175] Strains with single-prong shutoffs:

[0176] ZMY62 (TSM1 Ub only)

[0177] ZMY63 (TAF90 ub only)

[0178] ZMY64 (AF130 ub only)

[0179] HIS3 rox-only KO strain

[0180] The TAFs were shut-off by expressing each under direct control ofthe GAL 1,10 UAS+switching to glucose and by using the Ace-HIS3 (Kleinand Struhl) promoter, growing in the presence of copper and thenshifting by removing the copper (this, however, is less efficient thanwith galactose—cell growth never actually stopped, only slowed.)

[0181] The ub-only prong was tested by using a reporter plasmid(obtained from Dan Finley) called pUB23L (incorporating a Leu as theN-end residue in front of a ub-b-gal reporter. In the presence of 200 μMcopper and X-gal, the ub-only strain ZMY59 with this reporter is white.Without copper, it is blue.

[0182] A HIS3 DKO and a HIS3 Rox-only strain was tested and found thatin the presence of 5001M CuSO₄, both failed to grow on plates lackinghistidine. Without copper, growth was entirely normal on plates lackinghistidine.

[0183] The urlf allele is usually introduced by means of a one-stepintegration that simultaneously truncates the endogenous copy. (Thiscould also be done by shuffling in a cen plasmid in a null strain). Thestrain ZMY76) can stably integrate the urlf allele with a 2-step genereplacement. This means that there is no truncated piece left upstream,and no remaining repeated sequence fragments requiring maintenance ofselection for the integrating plasmid marker. Testing the system: Growthof the indicated strains (+ = strong growth; − = no growth; +/− =intermediate growth; ND = not determined). Allele 100 μM Cu 250 μM Cu500 μM Cu 1 mM ub-only TAF90 + ND − ND DKO TAF90 +/− − − − ub-onlyTAF130 + + + ND DKO TAF130 +/− − − − DKO TFIIB ND − − − ub-onlyTSM1 + + + + DKO TSM1 + + +/− −

[0184] Lee and Lis (1998) have used the system to shut off SRB4 andKIN28 (Nature 393:389-92)

[0185] I have used the system to shut off TOA1 (loss of much PolIItx-manuscript in prep), TAFs17, 40 and 67 (submitted 17 causes a generalloss of tx, and 40 and 67 cause gene-specific defects), and TAF23.

[0186] Equivalents

[0187] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. Such equivalents are considered tobe within the scope of this invention and are covered by the followingclaims.

We claim:
 1. A method for inducibly repressing the transcription of atarget gene expressed from a repressible promoter comprising: providingsaid target gene expressed from said repressible promoter, providing atranscriptional repressor expressed from an inducible promoter, whichtranscriptional repressor represses said repressible promoter, andproviding an inducing agent that induces said inducible promoter inorder to express said transcriptional repressor and repress saidrepressible promoter, thereby causing the transcriptional repression ofsaid target gene.
 2. The method of claim 1, wherein the target geneencodes a chimeric polypeptide comprising ubiquitin and a targetpolypeptide.
 3. The method of claim 1, wherein the repressible promoteris a promoter from a gene selected from the group consisting of ANB1,HEM13, ERG 11, OLE 1, GAL1, GAL10, and TET^(R).
 4. The method of claim1, wherein the transcriptional repressor is selected from the groupconsisting of ROX1, Tet repressor, and lacI repressor.
 5. The method ofclaim 1, wherein the inducible promoter is a copper-inducible promoter.6. The method of claim 1, wherein the inducible promoter is induced byCu⁺², tetracycline, or a gratuitous inducer of the lac operon.
 7. Amethod for inducibly degrading a target polypeptide wherein said targetpolypeptide is provided as a ubiquitin-target polypeptide fusion proteinin which a specific amino terminal amino acid residue of said targetpolypeptide is contiguously joined by a peptide bond to a carboxylterminal residue of a ubiquitin polypeptide, said method comprising:providing said ubiquitin-target polypeptide fusion protein, providing aubiquitin isopeptidase which endoproteolytically cleaves said peptidebond thereby liberating said specific amino terminal amino acid residueof said target polypeptide from said carboxyl terminal residue of saidubiquitin polypeptide, providing an inducible means for the proteolyticdestruction of said target polypeptide possessing said liberated aminoterminal amino acid residue, and inducing said inducible proteolyticmeans thereby causing the inducible proteolytic destruction of saidtarget polypeptide possessing said liberated amino terminal amino acidresidue.
 8. The method of claim 7, wherein the target polypeptide is aTAF.
 9. The method of claim 7, wherein the specific amino terminal aminoacid residue of said target polypeptide is arginine.
 10. The method ofclaim 7, wherein the specific amino terminal amino acid residue of saidtarget polypeptide is selected from the group consisting of arginine,lysine and histidine.
 11. The method of claim 7, wherein the specificamino terminal amino acid residue of said target polypeptide is selectedfrom the group consisting of phenylalanine, tryptophan, tyrosine,leucine, and isoleucine.
 12. The method of claim 7, wherein the specificamino terminal amino acid residue of said target polypeptide is selectedfrom the group consisting of aspartate, glutamate, cysteine, asparagineand glutamine.
 13. The method of claim 12, wherein the inducible meansfor the proteolytic destruction of the target polypeptide is aninducible transgene encoding an R-transferase.
 14. The method of claim12, wherein the specific amino terminal amino acid residue of saidtarget polypeptide is glutamine or asparagine and the inducible meansfor the proteolytic destruction of the target polypeptide is aninducible transgene encoding a deamidase specific for an amino-terminalglutamine or asparagine.
 15. The method of claim 7, wherein saidinducible proteolytic means is an inducible transgene encoding acomponent of the N-end rule system for ubiquitin dependent proteolyticdestruction.
 16. The method of claim 15, wherein said component of theN-end rule system is selected from the group consisting of UBR1, UBC2,NTA1, and ATE1.
 17. The method of claim 15, wherein said component ofthe N-end rule system is selected from the group consisting of mouseUBR1p and human UBR1p.
 18. A method for repressing the function of atarget gene expressing a target polypeptide by repressing thetranscription of said target gene and repressing the stability of saidtarget polypeptide, said method comprising: inducibly repressing thetranscription of said target gene by the method of claim 1, or induciblyrepressing the stability of said target polypeptide by the method ofclaim 7 thereby repressing the function of said target gene byrepressing the transcription of said target gene and repressing thestability of said target polypeptide.
 19. A method for repressing thefunction of a target gene expressing a target polypeptide by repressingthe transcription of said target gene and degrading said targetpolypeptide, said method comprising: inducibly repressing thetranscription of said target gene, and inducibly degrading said targetpolypeptide by the method of claim 7 thereby repressing the function ofsaid target gene by repressing the transcription of said target gene anddegrading said target polypeptide.
 20. A method for repressing thefunction of a target gene expressing a target polypeptide by repressingthe transcription of said target gene and degrading said targetpolypeptide, said method comprising: inducibly repressing thetranscription of said target gene, and inducibly degrading said targetpolypeptide thereby repressing the function of said target gene byrepressing the transcription of said target gene and degrading saidtarget polypeptide.
 21. A eukaryotic cell containing: a target geneencoding a target polypeptide an inducible transgene encodingproteolytic means for the degradation of a target polypeptide encoded bya target gene.
 22. The eukaryotic cell of claim 21, wherein saidinducible transgene comprises a component of the N-end rule proteolyticsystem.
 23. The eukaryotic cell of claim 22, wherein said component ofthe N-end rule proteolytic system is selected from the group consistingof UBR1, UBC2 and NTA1.
 24. The eukaryotic cell of claim 21, in whichsaid target gene is expressed from a repressible promoter.
 25. Theeukaryotic cell of claim 24, further containing an inducibletranscriptional repressor, which transcriptional repressor repressessaid repressible promoter.
 26. The eukaryotic cell of claim 25, whereinsaid repressible promoter is a promoter from a gene selected from thegroup consisting of ANB1, HEM13, ERG11, OLE1, GAL1, GAL10, and TET^(R).27. The eukaryotic cell of claim 25, wherein said transcriptionalrepressor is selected from the group consisting of ROX1, Tet repressor,and lacI repressor.
 28. A target gene chimera comprising a ubiquitinpolypeptide and a target polypeptide.
 29. The target gene chimera ofclaim 28, further comprising a specific amino terminal amino acidresidue of said target polypeptide wherein said specific amino terminalamino acid residue is contiguously joined by a peptide bond to acarboxyl terminal reside of said ubiquitin polypeptide.
 30. The targetgene chimera of claim 29, in which said specific amino terminal aminoacid residue is arginine.
 31. The target gene chimera of claim 29, inwhich said specific amino terminal amino acid residue is selected fromthe group consisting of arginine, lysine and histidine.
 32. The targetgene chimera of claim 29, in which said specific amino terminal aminoacid residue is selected from the group consisting of phenylalanine,tryptophan, tyrosine, leucine, and isoleucine.
 33. The target genechimera of claim 29, in which said specific amino terminal amino acidresidue is selected from the group consisting of aspartate, glutamate,cysteine, asparagine and glutamine.
 34. The target gene chimera of claim29, further comprising an epitope tag.
 35. The target gene chimera ofclaim 29, further comprising a segment of the lacI repressor carboxyl tosaid specific amino terminal amino acid residue.
 36. The target genechimera of claim 29, further comprising a lysine residue carboxyl tosaid specific amino terminal amino acid residue.
 37. A eukaryotic cellexpressing the target gene chimera of claim
 29. 38. The target genechimera of claim 29, wherein said target polypeptide is atranscriptional repressor which represses an inducible promoter.
 39. Themethod of claim 1, wherein the inducible promoter is repressed by aderepressible repressor and wherein said derepressible repressor isfurther subject to targeted proteolysis by the N-end rule system.